The purpose of the study was to demonstrate the feasibility of using transdermal iontophoresis to deliver a functional protein, ribonuclease A (RNAse; 13.6 kDa), non-invasively across the skin. Iontophoretic transport experiments were conducted using porcine skin in vitro and established the effect of current density and protein concentration on delivery kinetics. A methylene blue-based assay was used to quantify RNAse transport and to simultaneously demonstrate that protein functionality was retained post-iontophoresis. The results confirmed that intact functional RNAse was indeed delivered across the skin; cumulative permeation and steady state flux after 8h iontophoresis at 0.3 mA/cm(2) were 224.37+/-72.34 microg/cm(2) and 68.28+/-23.87 microg/cm(2)h, respectively. Significant amounts of protein were also deposited within the membrane (e.g., 1425.13+/-312.09 microg/cm(2) at 0.3 mA/cm(2)). In addition to the evidence provided by the enzymatic assay with regards to RNAse integrity and functionality, SDS-PAGE gels and MALDI-TOF spectra were also used to characterize RNAse present in the receiver phase (MALDI-TOF spectra: RNAse control, 13.690 kDa cf. RNAse from permeation samples, 13.692 kDa). Co-iontophoresis of acetaminophen showed that, despite its molecular weight, electromigration was the predominant electrotransport mechanism, accounting for >80% of RNAse total flux. Increasing RNAse concentration from 0.35 to 0.7 mM in the formulation did not result in a statistically significant increase in delivery. Iontophoretic transport of RNAse across human skin was statistically equivalent to that seen with porcine skin under the same conditions; cumulative permeation across human and porcine skin was 241.48+/-60.01 and 170.71+/-92.13 microg/cm(2), respectively. Laser scanning confocal microscopy was used to visualize the distribution of rhodamine B-labelled RNAse in the epidermis and dermis as a function of depth following 8h iontophoresis (results were compared to control experiments involving passive administration of the same formulation for 8h). Although fluorescence was localized at the skin surface following passive administration, it was visible throughout the membrane after current application. In conclusion, the results demonstrate that non-invasive transdermal iontophoresis can be used to deliver significant amounts of a structurally intact, functional protein across skin.