Elevated expression levels of the bcl-2 proto-oncogene have been extensively correlated with the appearance of androgen independence in prostate cancer. Although bcl-2 was first cloned as the t(14:18) translocation breakpoint from human follicular B cell lymphoma, the mechanism of overexpression of bcl-2 is largely undefined for advanced prostate cancer because there are no gross alterations in the gene structure. We investigated the role of the product of the prostate apoptosis response gene-4 (Par-4) and the product of the Wilms' tumor 1 gene (WT1) in the regulation of Bcl-2 expression in prostate cancer cell lines. We observed growth arrest and apoptosis, upon decreasing Bcl-2 protein and transcript in the high Bcl-2-expressing, androgen-independent prostate cancer cell line, by all-trans-retinoic acid treatment (ATRA), but this did not occur in the androgen-dependent cell line expressing low levels of Bcl-2. The decrease in the Bcl-2 protein and transcript following all-trans-retinoic acid treatment was accompanied by changes in localization of Par-4 and an induction in the expression of WT1 protein. In stable clones expressing ectopic Par-4 and in ATRA-treated cells, we observed decreased Bcl-2 protein and transcript. This was accompanied by an induction in WT1 expression. The involvement of WT1 in the Par-4-mediated down-modulation of Bcl-2 was further defined by blocking endogenous WT1 expression, which resulted in an increase in Bcl-2 expression. Finally, we detected Par-4 and WT1 proteins binding to a previously identified WT1-binding site on the bcl-2 promoter both in vitro and in vivo leading to a decrease in transcription from the bcl-2 promoter. We conclude that Par-4 regulates Bcl-2 through a WT1-binding site on the bcl-2 promoter. These data also identify Par-4 nuclear localization as a novel mechanism for ATRA-mediated bcl-2 regulation.