VIP receptors are frequently overexpressed by various endocrine tumors. In this study the expression of VIP receptors in the human adrenocortical carcinoma cell line NCI-H295 and their involvement in the regulation of steroidogenesis was investigated. NCI-H295 cells express VIP1 and VIP2 receptors as demonstrated by RT-PCR, whereas they do not express VIP itself. The receptors are functionally coupled to steroidogenesis since VIP (10(-9) M to 10(-6) M) exerted a dose-dependent stimulatory effect on the release of aldosterone, cortisol, and DHEA. VIP increased ACTH-stimulated releases of aldosterone and cortisol. The proliferation rate of NCI-H295 cells was not affected by VIP. These data show that NCI-H295 cells express both forms of the VIP receptor and that VIP is involved in an ACTH-independent regulation of steroidogenesis in the adrenal tumor cells.