Polychlorinated biphenyls (PCBs) are complex mixtures of congeners that exhibit carcinogenic and toxicant activities in a variety of mammalian tissues. Here, we studied the acute in vivo and in vitro effects of a commercially used PCB product, Aroclor 1248 (A1248), a mixture of tri-, tetra-, and pentachloro congeners. Single intraperitoneal (i.p.) or bilateral intratesticular (i.t.) injections of A1248 decreased serum androgen levels in both groups 24 h after injection. Chorionic gonadotropin–stimulated androgen production by acute testicular cultures from both groups was also reduced, and progesterone production was attenuated in cultures from i.t.-treated animals. The capacity of the postmitochondrial fractions from testes of i.t.-treated animals to convert pregnenolone to progesterone and progesterone to testosterone was reduced as well. In vitro studies revealed that a 10- to 15-min exposure of postmitochondrial testicular fractions and intact interstitial cells from normal animals to A1248 in a subnanomolar concentration range was sufficient to attenuate the conversion of pregnenolone to progesterone and progesterone to testosterone. At micromolar concentrations, A1248 added in vitro also inhibited the conversion of Δ⁴-androstendione to testosterone without affecting the viability of interstitial cells. These results indicate that A1248 down-regulates the testicular androgenesis by an acute inhibition of 3β-hydroxysteroid dehydrogenase, 17α-hydroxylase/lyase, and 17β-hydroxysteroid dehydrogenase activities.