Plasma-membrane carboxypeptidase-D (CPD) releases arginine from extracellular substrates. Arginine is converted intracellularly to nitric oxide (NO). This study determined the effects of testosterone (T) and prolactin (PRL) on CPD expression, and the role(s) of CPD in NO production and survival of prostate cancer (PCa) cells. LNCaP cells were treated with T and/or PRL. CPD expression was measured. Regulation by T (low doses) was determined using transfected cells overexpressing 5-reductase type-1 (5R1), which converts T to the more potent dihydrotestosterone. The effects of siRNAs targeting CPD (siCPDs) on NO production, cell viability, and apoptosis were determined using DAF2-DA, MTS, and Annexin-V assays. The effects of PRL/T on CPD/NO levels in PC-3, MDA-PCa-2b, and 22Rv1 cells were also evaluated. In LNCaP cells, 10nM T and 10ng/ml PRL-upregulated CPD mRNA/protein levels. In pTRE-transfectants, 1nM T-upregulated CPD mRNA levels by 2-fold over controls, whereas 0.1nM T caused similar upregulation in pTRE-5R1-transfectants. In LNCaP cells cultured in arginine-free medium, addition of furylacryloyl-Ala-Arg (FAR; CPD substrate) increased NO levels. NO production, with FAR, was enhanced by PRL and/or T. siCPDs decreased NO production and cell viability, but increased apoptosis. QPCR analysis showed T/PRL-upregulation of CPD in 22Rv1, MDA-PCa-2b, and PC-3 cells. NO production was doubled by T/PRL in 22Rv1 cells, tripled by T in MDA-PCa-2b cells, and marginally increased by PRL in MDA-PCa-2b and PC-3 cells. T and PRL upregulate CPD and NO levels in PCa cells. CPD increases NO production to promote PCa cell survival.