PURPOSE: SPECT imaging with two radiotracers at the same time is feasible if two different radioisotopes are employed, given their distinct energy emission spectra. In the case of (123)I and (125)I, dual SPECT imaging is not straightforward: (123)I emits photons at a principal energy emission spectrum of 143.1-179.9keV. However, it also emits at a secondary energy spectrum (15-45keV) that overlaps with the one of (125)I and the resulting cross-talk of emissions impedes the accurate quantification of (125)I. In this paper, we describe three different methods for the correction of this cross-talk and the simultaneous in vivo [(123)I]IBZM and [(125)I]R91150 imaging of D2/3 and 5-HT2A receptors in the rat brain. METHODS: Three methods were evaluated for the correction of the effect of cross-talk in a series of simultaneous, [(123)I]IBZM and [(125)I]R91150 in vivo and phantom SPECT scans. Method 1 employs a dual-energy window (DEW) approach, in which the cross-talk on (125)I is considered a stable fraction of the energy emitted from (123)I at the principal emission spectrum. The coefficient describing the relationship between the emission of (123)I at the principal and the secondary spectrum was estimated from a series of single-radiotracer [(123)I]IBZM SPECT studies. In Method 2, spectral factor analysis (FA) is applied to separate the radioactivity from (123)I and (125)I on the basis of their distinct emission patterns across the energy spectrum. Method 3 uses a modified simplified reference tissue model (SRTMC) to describe the kinetics of [(125)I]R91150. It includes the coefficient describing the cross-talk on (125)I from (123)I in the model parameters. The results of the correction of cross-talk on [(125)I]R91150 binding potential (BPND) with each of the three methods, using cerebellum as the reference region, were validated against the results of a series of single-radiotracer [(123)I]R91150 SPECT studies. In addition, the DEW approach (Method 1), considered to be the most straightforward to apply of the three, was further applied in a dual-radiotracer SPECT study of the relationship between D2/3 and 5-HT2A receptor binding in the striatum, both at the voxel and at the regional level. RESULTS: Average regional BPND values of [(125)I]R91150, estimated on the cross-talk corrected dual-radiotracer SPECT studies provided satisfactory correlations with the BPND values for [(123)I]R91150 from single-radiotracer studies: r=0.92, p<0.001 for Method 1, r=0.92, p<0.001 for Method 2, r=0.92, p<0.001, for Method 3. The coefficient describing the ratio of the (123)I-emitted radioactivity at the (125)I-emission spectrum to the radioactivity that it emits at its principal emission spectrum was 0.34 in vivo. Dual-radiotracer in vivo SPECT studies corrected with Method 1 demonstrated a positive correlation between D2/3 and 5-HT2A receptor binding in the rat nucleus accumbens at the voxel level. At the VOI-level, a positive correlation was confirmed in the same region (r=0.78, p<0.01). CONCLUSION: Dual-radiotracer SPECT imaging using (123)I and (125)I-labeled radiotracers is feasible if the cross-talk of (123)I on the (125)I emission spectrum is properly corrected. The most straightforward approach is Method 1, in which a fraction (34%) of the radioactivity emitted from (123)I at its principal energy spectrum is subtracted from the measured radioactivity at the spectrum of (125)I. With this method, a positive correlation between the binding of [(123)I]IBZM and [(125)I]R91150 was demonstrated in the rat nucleus accumbens. This result highlights the interest of dual-radiotracer SPECT imaging to study multiple neurotransmitter systems at the same time and under the same biological conditions.