Postnatal development of Leydig cells involves transformation through three stages: progenitor, immature, and adult Leydig cells. The process of differentiation is accompanied by a progressive increase in the capacity of Leydig cells to produce testosterone (T). T promotes the male phenotype in the prepubertal period and maintains sexual function in adulthood; therefore, disruption of T biosynthesis in Leydig cells can adversely affect male fertility. The present study was designed to evaluate the ability of a xenoestrogen, methoxychlor (the methoxylated isomer of DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane]), to alter Leydig cell steroidogenic function. Purified progenitor, immature, and adult Leydig cells were obtained from, respectively, 21-, 35-, and 90-day-old Sprague-Dawley rats treated with graded concentrations of the biologically active metabolite of methoxychlor, 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), and assessed for T production. HPTE caused a dose-dependent inhibition of basal and LH-stimulated T production by Leydig cells. Compared to the control value, reduced T production by progenitor and immature Leydig cells was apparent after 10 h of HPTE treatment in culture; the equivalent time for adult Leydig cells was 18 h. The reversibility of HPTE-induced inhibition was evaluated by incubating Leydig cells for 3, 6, 10, 14, or 18 h and measuring T production after allowing time for recovery. After treatment with HPTE for 3 h, T production by immature and adult Leydig cells for the 18-h posttreatment period was similar to the control value, but that of progenitor Leydig cells was significantly lower. The onset of HPTE action and the reversibility of its effect showed that Leydig cells are more sensitive to this compound during pubertal differentiation than in adulthood. T production was comparable when control and HPTE-treated immature Leydig cells were incubated with pregnenolone, progesterone, and androstenedione, but HPTE-treated Leydig cells produced significantly reduced amounts of T when incubations were conducted with 22R-hydroxycholesterol (P < 0.01). This finding suggested that HPTE-induced inhibition of T production is related to a decrease in the activity of cytochrome P450 cholesterol side-chain cleavage enzyme (P450_scc) and cholesterol utilization. The reduced steady-state mRNA level for P450_scc in HPTE-treated Leydig cells was demonstrated by reverse transcription-polymerase chain reaction and densitometry. In conclusion, this study showed that HPTE causes a direct inhibition of T biosynthesis by Leydig cells at all stages of development. This effect suggests that reduced T production could be a contributory factor in male infertility associated with methoxychlor and, possibly, other DDT-related compounds.