We have previously shown in B cell lines that the cAMP-response element (CRE) is a major positive regulatory site in the bcl-2 promoter. This element is not only essential for bcl-2 deregulation in t(14;18) cells, but it is also responsible for the positive regulation of bcl-2 expression during the activation of mature B cells and the rescue of immature B cells from calcium-dependent apoptosis in vitro. However, the role of the CRE in the regulation of endogenous bcl-2 expression in vivo has not been characterized. We used gene targeting to generate knock-in mice in which a mutant CRE site was introduced into the bcl-2 promoter region. The mutant CRE reduced the expression of bcl-2 mRNA in several tissues, including thymus, kidney, lung, liver, brain and heart. The levels of bcl-2 mRNA and protein were also significantly lower in splenic B cells from the knock-in mice. Consistent with these results, the activation of B cells from the knock-in mice by anti-CD 40, lipopolysaccharide (LPS) or anti-IgM was reduced as compared to B cells from wild-type littermates. B cells with the mutant CRE were more susceptible to the induction of apoptosis with several different agents consistent with the decreased expression of bcl-2. Preliminary flow cytometric studies suggest that the number of B cells is decreased in the knock-in mice at 8 weeks of age. Quantitative chromatin immunoprecipitation assays revealed essentially no binding of CREB or ATF-2 and decreased binding of CBP and c-Rel to the mutant CRE site in the bcl-2 promoter. Our previous studies have shown that the CRE site in the bcl-2 promoter is linked to the mediation of signal transduction pathways in B cells, so we investigated the effect of forskolin, a cAMP-elevating agent. We found that treatment of the B cells from the knock-in mice with forskolin led to significantly more cell death than observed with wild-type B cells. Taken together, these findings indicate that the CRE site in the bcl-2 promoter has a functional role in the regulation of endogenous bcl-2 expression and plays an important role in the regulation of apoptosis in B cells.