The bovine gene for the steroidogenic acute regulatory protein (StAR) was cloned and sequenced, including 2 kb of the upstream control region of the gene. The gene comprises seven exons arranged similarly to those of the human and mouse gene sequences. The sequence analysis identified three cis elements corresponding to the binding motif for the transcription factor SF-1/Ad4BP, at - 100, - 240 and - 1190 from the transcription start site. Electrophoretic mobility shift analysis (EMSA) using nuclear proteins from bovine corpus luteum and bovine adrenal as well as in vitro transcribed/translated SF-1/Ad4BP consistently showed that only the site at -1190 bound the transcription factor significantly. Very weak binding was detectable also at the - 240 site, but none at the -100 site. Heterologous transfection of StAR promoter deletion-reporter constructs into Hela cells cotransfected with an expression vector for bovine SF-1/Ad4BP, showed that this transcription factor can specifically act on the bovine StAR gene promoter, but preferentially in regions corresponding to the two proximal SF-1/Ad4BP elements at - 100 and - 240, though with only low relative effect. Furthermore, additional cotransfection of a construct expressing a constitutive protein kinase A catalytic subunit to mimic the effects of cAMP stimulation, led to a small SF-1/Ad4BP-dependent increase in reporter activity mediated only by the same proximal sites. Since the bovine StAR gene promoter does not appear to have a functional cAMP responsive element (CRE), either this effect is mediated in this system directly by SF-1/Ad4BP, or by other factors interacting with this transcription factor, but which do not involve CRE-mediated gene activation. Taken together, the results show that there is a discrepancy between the results of the EMSA experiments and those using transfection of promoter-reporter constructs, which needs to be resolved before a clear understanding of SF-1/Ad4BP-mediated regulation of the StAR gene is attained.