PURPOSE: Accurate and robust image segmentation was identified as one of the most challenging issues facing PET quantification in oncological imaging. This difficulty is compounded by the low spatial resolution and high noise characteristics of PET images. The fuzzy C-means (FCM) clustering algorithm was largely used in various medical image segmentation approaches. However, the algorithm is sensitive to both noise and intensity heterogeneity since it does not take into account spatial contextual information. METHODS: To overcome this limitation, a new fuzzy segmentation technique adapted to typical noisy and low resolution oncological PET data is proposed. PET images smoothed using a nonlinear anisotropic diffusion filter are added as a second input to the proposed FCM algorithm to incorporate spatial information (FCM-S). In addition, a methodology was developed to integrate the a trous wavelet transform in the standard FCM algorithm (FCM-SW) to allow handling of heterogeneous lesions' uptake. The algorithm was applied to the simulated data of the NCAT phantom, incorporating heterogeneous lesions in the lung and clinical PET/CT images of 21 patients presenting with histologically proven nonsmall-cell lung cancer (NSCLC) and 7 patients presenting with laryngeal squamous cell carcinoma (LSCC) to assess its performance for segmenting tumors with arbitrary size, shape, and tracer uptake. For NSCLC patients, the maximal tumor diameters measured from the macroscopic examination of the surgical specimen served as the ground truth for comparison with the maximum diameter estimated by the segmentation technique, whereas for LSCC patients, the 3D macroscopic tumor volume was considered as the ground truth for comparison with the corresponding PET-based volume. The proposed algorithm was also compared to the classical FCM segmentation technique. RESULTS: There is a good correlation (R2 = 0.942) between the actual maximal diameter of primary NSCLC tumors estimated using the proposed PET segmentation procedure and those measured from the macroscopic examination, and the regression line agreed well with the line of identity (slope = 1.08) for the group analysis of the clinical data. The standard FCM algorithm seems to underestimate actual maximal diameters of the clinical data, resulting in a mean error of -4.6 mm (relative error of -10.8 +/- 23.1%) for all data sets. The mean error of maximal diameter estimation was reduced to 0.1 mm (0.9 +/- 14.4%) using the proposed FCM-SW algorithm. Likewise, the mean relative error on the estimated volume for LSCC patients was reduced from 21.7 +/- 22.0% for FCM to 8.6 +/- 28.3% using the proposed FCM-SW technique. CONCLUSIONS: A novel unsupervised PET image segmentation technique that allows the quantification of lesions in the presence of heterogeneity of tracer uptake was developed and evaluated. The technique is being further refined and assessed in clinical setting to delineate treatment volumes for the purpose of PET-guided radiation therapy treatment planning but could find other applications in clinical oncology such as the assessment of response to treatment.