BACKGROUND Recent data has shown that prostate cancer (PCA) cells are capable of producing testosterone directly from cholesterol, which may contribute to the development of castration resistance. While up-regulation of steroidogenic enzymes has been previously described during castration-resistant prostate cancer (CRPC) progression, regulation of this process is poorly defined. These data examine the role of luteinizing hormone (LH) in the regulation of steroidogenic machinery in PCA cells. METHODS PCA cell lines LNCaP, C4-2B, and 22RV1 were exposed to LH. Gene expression was quantified using real-time PCR and protein expression was characterized with standard Western blot analysis. Steroid analysis was performed using radioimmunoassay (RIA). Cell viability was measured using an MTS viability assay. RESULTS Androgen-sensitive (LNCaP) and -independent PCA cells (C4-2B and 22RV1) express both mRNA and protein for LH and LH receptor (LHR). Exposure of these cells to LH for 4 hr increased the expression of several steroidogenic genes. Exposure for 10 days resulted in the increase of additional genes. At both time points, the upregulation of these genes was dose-dependent. This was mirrored by an increase in the expression of several key steroidogenic enzymes, including StAR, CYB5B, CYP11A, and 3βHSD. LH stimulated the production of progesterone and testosterone in LNCaP cells as measured by RIA. We have also demonstrated that treatment of LNCaP cells with LH enhanced their viability. CONCLUSIONS Our data show that LH-mediated activation of LHR significantly up-regulates the expression of genes and enzymes required for steroidogenesis and increases steroid production in PCA cells.