The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) that is responsible for detoxifying isocaproaldehyde generated by steroidogenesis. In adrenocortical cell cultures, hormonal regulation of MVDP gene occurs through the cAMP pathway. We show that in adrenals, the pituitary hormone ACTH regulates MVDP gene expression in a coordinate fashion with steroidogenic genes. Cell transfection and DNA-binding studies were used to investigate the molecular mechanisms underlying MVDP gene regulation in Y1 adrenocortical cells. Progressive deletions of upstream regulatory regions identified a -121/+41 fragment that was sufficient for basal and cAMP-mediated transcriptional activities. Gel shift assays showed that CTF1/nuclear factor 1 (NF1), CCAAT enhancer binding protein-ss (C/EBPss), and selective promoter factor 1 (Sp1) factors bound to cis-acting elements at positions -76, -61, and -52, respectively. We report that the cell-specific steroidogenic factor-1 (SF-1) interacts specifically with a novel regulatory element located in the downstream half-site of the proximal androgen response element (AREp) at position -102. Functional analysis of SF-1 and NF1 sites in the -121/+41 promoter showed that mutation of one of them decreases both constitutive and forskolin-stimulated promoter activity without affecting the fold induction (forskolin stimulated/basal). Individual mutations of C/EBP and Sp1 sites resulted in a loss of more than 50% of the cAMP-dependent induction. When both sites were mutated simultaneously, cAMP responsiveness was nearly abolished. Thus, in adrenocortical cells, both SF-1 and NF1 are required for high expression of the MVDP promoter while Sp1 and C/EBPss functionally interact in an additive manner to mediate cAMP-dependent regulation. Furthermore, we report that MVDP gene regulation is impaired in stably transfected Y1 clones expressing DAX-1. Taken together, our findings suggest that detoxifying enzymes of the aldose reductase family may constitute new potential targets for regulators of adrenal and gonadal differentiation and function, e.g. SF-1 and DAX-1.