In this study, we hypothesized that many of the reported effects of phthalate esters and other peroxisome proliferators (PPs) in the testis are mediated by members of the PP- activated receptor (PPAR) family of transcription factors through alterations in proteins involved in steroidogenesis. Exposure of Leydig cells to PPs prevented cholesterol transport into the mitochondria after hormonal stimulation and inhibited steroid synthesis, without altering total cell protein synthesis or mitochondrial and DNA integrity. PPs also reduced the levels of the cholesterol-binding protein peripheral-type benzodiazepine receptor (PBR) because of a direct transcriptional inhibition of PBR gene expression in MA-10 Leydig cells. MA-10 cells contain mRNAs for PPARalpha and PPARbeta/delta, but not for PPARgamma. In vivo treatment of mice with PPs resulted in the reduction of both testis PBR mRNA and circulating testosterone levels, in agreement with the proposed role of PBR in steroidogenesis. By contrast, liver PBR mRNA levels were increased, in agreement with the proposed role of PBR in cell growth/tumor formation in nonsteroidogenic tissues. However, PPs did not inhibit testosterone production and testis PBR expression in PPARalpha-null mice. These results suggest that the antiandrogenic effect of PPs is mediated by a PPARalpha-dependent inhibition of Leydig cell PBR gene expression.