An improved technique for cytogenetic analysis of malignant prostatic tissue is described. This method is based on 1) prolonged mild collagenase treatment, 2) careful washing and repeated centrifugation and sedimentation of the disaggregated material to isolate viable prostatic epithelial cells, 3) short-term culture on collagen R-coated chamber slides with PFMR-4 medium supplemented with mitogenic factors, and 4) daily inspection of the cultured cells to determine the optimal time for harvesting. Twenty consecutive primary prostatic adenocarcinomas were cultured and processed for cytogenetic analysis. Outgrowth of pure epithelial colonies was obtained in 16 cases; in three there was a mixture of epithelial colonies and fibroblasts, and in one there was no cell growth. More than 25 metaphases with chromosomes of high banding quality could be analyzed per case, in particular in cultures from the final pellet fraction. Clonal chromosome aberrations were present in four tumors, and nonclonal structural changes were present in nine. Six tumors showed only normal diploid karyotypes.