Transcription of the rat CYP24 gene is induced by 1, 25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) through two vitamin D response elements (VDREs). A functional Ras-dependent Ets-binding site (EBS) was located downstream from the proximal VDRE and was critical to 1,25(OH)(2)D(3)-mediated induction. Cotransfection of Ets-1 and Ets-2 stimulated induction, which was lost when the EBS was mutated. Multiple nuclear-protein complexes from COS-1 cells bound to the EBS in which three complexes were immunologically related to Ets-1. Transcriptional synergy was observed between the proximal VDRE and adjacent EBS as was the attendant formation of a ternary complex between vitamin D receptor- retinoid X receptor (VDR. RXR) and Ets-1. In the absence of 1,25-(OH)(2)D(3) or in the presence of an inactive proximal VDRE, the EBS failed to respond to exogenous Ets-1. However, Ets-1 increased basal expression when cotransfected with a mutant VDR. The inductive action of 1, 25-(OH)(2)D(3) was substantially increased by Ras, which was ablated by mutagenesis of the EBS or by expression of a mutated Ets-1 protein (T38A). EBS contribution to hormone induction was prevented by manumycin A, an inhibitor of Ras farnesylation. A fundamental role was established for transcriptional cooperation between Ras-activated Ets proteins and the VDR.RXR complex in mediating 1, 25-(OH)(2)D(3) action on the CYP24 promoter.