Leydig cells of the mammalian testis produce testosterone and support spermatogenesis, and thereby their role in male function is fundamental. Although benzo[a]pyrene (B[a]P) has been known to exhibit carcinogenic, apoptogenic, and endocrine disrupting activities, its potential signaling system in Leydig cells remain to be discovered. In the present study, using the TM3 Leydig cell line and primary Leydig cells, we showed that Leydig cells do not die by exposure to B[a]P, and found that an increased level of XIAP may be associated with the anti-apoptotic process. The Leydig cells were shown to express p53 but its translational level was extremely low. Although a high level of p53 protein was not necessary for BPDE (a final B[a]P metabolite)-induced apoptosis in Leydig cells, the apoptosis of primary Leydig cells appears to be p53-independent. This indicates the lack of p53 function in primary Leydig cells. Furthermore, Leydig cells were found to retain insignificant levels of endogenous AhR and Arnt proteins in nature. Exposure to B[a]P did not result in a significant increase in AhR proteins that are required for CYP1A1 transcription. CYP1A1 expression was present in Leydig cells but at levels insufficient to exhibit its activity. Finally, we have demonstrated that overexpression of CYP1A1 in Leydig cells sensitizes the cells to exhibit its activity in the presence of B[a]P, and thus induction of apoptosis. Together, these results indicate that the deficiency of CYP1A1 activity might be a decisive condition rendering Leydig cells secure from exogenous PAHs like B[a]P.