CREB-1 is expressed in the bone marrow and in developing B cells. To determine the role of CREB-1 in developing B cells in the bone marrow, several lines of transgenic (Tg) mice overexpressing a dominant-negative Ser(119-ala) phosphomutant CREB-1 in the bone marrow were generated. Analysis of RNA and protein revealed expression of the transgene in the bone marrow. Flow cytometric analysis of bone marrow cells from Tg mice revealed approximately 70% increase in pre-B1 (CD43(+)B220(+)CD24(+(int))) and approximately 60% decreased pre-BII (CD43(+)B220(+)CD24(++(high))) cells, indicating a developmental block in pre-BI to pre-BII transition. Consistent with this, the Tg mice showed approximately 4-fold decrease in immature and mature B cells in the bone marrow. RT-PCR analysis of RNA from Tg mice revealed increased JunB and c-Jun in pre-BII cells associated with decreased S-phase entry. Adoptive transfer of bone marrow cells into RAG-2(-/-) mice resulted in reconstitution of non-Tg but not Tg bone marrow-derived CD43(+)B220(+)CD24(high) population that is normally absent in RAG-2(-/-) mice. In the periphery, the Tg mice exhibited decreased CD21(dim)CD23(high)IgM(+) follicular B cells in the spleen and increased B1a and B1b B cells in the peritoneum. While exhibiting normal Ab responses to T-independent Ags and primary response to the T-dependent Ag DNP-keyhole limpet hemocyanin, the Tg mice exhibited severely impaired secondary Ab responses. These studies provide the first evidence for a differential role for CRE-binding proteins in multiple stages of B cell development, functional maturation, and B1 and B2 B cells.