A crucial event in the acute regulation of steroidogenesis by trophic hormones is the delivery of cholesterol into the mitochondria where it is converted to pregnenolone by the cholesterol side chain cleavage enzyme. Although considerable controversy exists regarding the exact mechanisms that underlie this acute response to hormone stimulation, recent studies suggest that the Steroidogenic Acute Regulatory (StAR) protein, a hormone-induced 30-kilodalton mitochondrial protein, plays an essential role. We now extend these studies by establishing in MA-10 mouse Leydig tumor cells a temporal relationship between levels of StAR expression and steroidogenesis in response to hormone stimulation. These data indicate that trophic hormones regulate StAR mRNA and protein within a time frame concomitant with the acute production of steroid hormones and provide the first evidence implicating changes in StAR transcription and/or mRNA stability in the functional response of steroidogenic cells to hormone action. In addition, in situ hybridization analyses of StAR expression in embryonic and adult mice demonstrated a precise spatial and temporal relationship in vivo between StAR expression and the capacity to produce steroid hormones. These experiments strengthen considerably the evidence that StAR is the key mediator of the acute induction of steroidogenesis and provide new insights into the mechanisms by which trophic hormones activate steroidogenesis in steroidogenic cells.