A key regulatory point in fine tuning of steroidogenesis is the synthesis of steroidogenic acute regulatory protein, which transfers cholesterol into mitochondria. Heat shock and toxic insults reduce steroidogenic acute regulatory protein, severely compromising steroid synthesis. As the molecular mechanisms for this reduction remain elusive, we tested the hypothesis that heat shock directly interferes with transcription of the steroidogenic acute regulatory protein gene. We show that, in mouse MA-10 Leydig tumor cells, heat shock caused drastic declines in (Bu)(2)cAMP-induced progesterone accumulation and steroidogenic acute regulatory protein transcript abundance. A proximal steroidogenic acute regulatory protein promoter fragment (-85 to +39) is sufficient to direct both cAMP inducibility and heat shock inhibition. Nuclear extracts from MA-10 cells displayed binding to this proximal promoter fragment as a low mobility complex in gel shift experiments. This complex disappeared in nuclear extracts taken at 5 and 10 min after initiation of heat shock and reappeared in extracts taken at 2 and 8 h. Similar low- mobility complexes formed on oligonucleotides representing the overlapping subfragments of the minimal steroidogenic acute regulatory protein promoter fragment sensitive to the heat shock effect. Extracts from heat-shocked MA-10 cells displayed reduced complex formation to each of the subfragments. We conclude that heat shock reduces progesterone synthesis, steroidogenic acute regulatory protein mRNA abundance, and steroidogenic acute regulatory protein promoter activity and disrupts binding of nuclear proteins to the proximal region of the steroidogenic acute regulatory protein promoter. Together these observations provide strong evidence for a mechanism of transcriptional inhibition in the down-regulation of steroidogenic acute regulatory protein expression by heat shock.