Castration-resistant prostate cancer (CRPC) is a major challenge for the development of efficient prostate cancer (PCa) therapies. Recent studies suggest that PCa progression to castration-resistant stage is associated with changes in intratumor androgen biosynthesis that would maintain the androgen-stimulated growth of the cancerous tissue. Thus, blocking intratumor androgen biosynthesis is an attractive option for drug development. We have applied the VCaP xenograft model for studying the castration-resistant stage of PCa. In this study, VCaP cells were inoculated orthotopically into the dorsolateral prostate of nude mice and tumor growth was followed by monitoring serum PSA concentrations. Tumor tissues were collected during the study at three different time points. When serum PSA reached 10 mg/l, a group of animals were castrated, causing tumors to regress, after which tumors were allowed to regrow into castration-resistant stage (PSA 10 mg/l). Tumor and serum samples were collected and concentrations of 10 steroids were analyzed by HPLC-MS/MS. Despite of the decrease in serum androgen concentrations after castration, no decline was observed in tumor androgen concentrations 3–7 weeks after castration. Interestingly, we observed significant changes in the expression of certain steroidogenic enzyme genes, such as HSD17B6 (2-fold, p=0.006) and AKR1C3 (2-fold, p=0.002). Furthermore, we detected up-regulation of androgen receptor (>15-fold, p<0.001) in the castration-resistant VCaP tumors. The results indicate that VCaP tumors grown in castrated mice produce significant levels of active androgens, independent of gonadal steroid synthesis. We suggest that enzymes involved in intratumor androgen production are attractive drug targets for the development of CRPC therapies.