The enzyme thimet oligopeptidase (TOP) is present in a wide variety of cell types and hydrolyzes many peptides of physiological importance in neuroendocrine pathways. TOP has been identified in prostate cancer cells, and its activity is increased by androgens in androgen‐responsive cells. Using the androgen‐sensitive cell line DU145‐AR, we have found that TOP levels and activity were significantly increased after 24 hours incubation with either dihydrotestosterone or estradiol. TOP was found both intracellularly and in the medium. These increases were not observed with an androgen‐insensitive line (DU145‐T). TOP works in concert with the peptidase prolyl endopeptidase (PEP) in breakdown of gonadotropin‐releasing hormone (GnRH), a peptide known to influence growth of prostate cells. We have identified PEP in DU145‐AR cells and seen an increase in PEP levels with androgen treatment. Furthermore, immunocytochemistry revealed that TOP was more clearly localized to nuclei at high levels of estradiol, compared to its distribution between cytoplasm and nucleus in the absence of steroid or at low levels of estradiol. These results suggest that peptidases, especially those involved in GnRH metabolism, may be involved in regulation of growth of prostate cancer cells and, hence, be targets for cancer therapy.