The oxidation and isomerization of 3beta-hydroxy-5-ene steroids into keto-4-ene steroids, a pivotal step in the synthesis of all hormonal steroids, is catalyzed by several isoforms of 3beta-hydroxysteroid dehydrogenase. In humans, two highly homologous isoforms exist, type I expressed by the HSD3B1 gene in peripheral tissues, and type II expressed by the HSD3B2 gene in steroidogenic organs. Previously, it was shown that the HSD3B1 gene 3beta1-A element, encompassing 24 nucleotides of intron 1 not perfectly conserved between the two genes and overlapping with a conserved TG box, contributes to maximal basal promoter activity by binding the ubiquitous and unidentified 3beta1-A transcription factor. In this study for the first time we report that similarly, the HSD3B2 gene intron 1 is required for maximal basal promoter activity in reporter gene analyses, as lack of intron 1 results in a 4- to 10-fold reduction in promoter activity. Mutational analysis in gel shift assays revealed that the 3beta1-A factor binds both the HSD3B2 and HSD3B1 gene intron 1 by requiring only seven nucleotides of a conserved segment within the 3beta1-A element. By competition analysis and use of anti-YY1 antibody in both gel shift and Western blot experiments, we identified the 3beta1-A protein as the ubiquitous transcription factor YY1. In addition, we have characterized another similar YY1 binding site differently located with respect to the 3beta1-A element in both genes. Deletion and mutational analysis in transient transfections experiments revealed that contrarily to as previously shown for the HSD3B1 gene, lack of YY1 binding to the type II 3beta1-A element only results in a marginal reduction of basal promoter activity. Instead, YY1 binding to the second site, placed 35 bp downstream from the 3beta1-A element, strongly activates the HSD3B2 gene basal promoter activity, as preventing YY1 binding to this region caused a 50% decrease of basal transcription. Complete abrogation of YY1 binding within type II intron 1 resulted in a gene reporter activity identical to a reporter construct lacking the whole intron 1. These results designate YY1 as the factor responsible for the intron 1-mediated boost of the HSD3B2 gene basal promoter activity. Similarities and dissimilarities between YY1 binding within the HSD3B1 and HSD3B2 gene intron 1 are discussed involving the conserved intron 1 TG box, that suggests different mechanisms are implicated in the YY1-mediated stimulation of these two genes basal promoter activity.