Immunoglobulin heavy chain (IgH) gene expression is guided by cis-regulatory elements which direct correct temporal and spatial expression in B lineage cells. One of these cis-acting elements is the IgH HS1,2 enhancer and previous studies in transgenic mice have revealed a temporally restricted activity of an HS1,2 enhancer-linked human beta-globin reporter gene in B lineage cells. To assess whether this enhancer can impose strict temporal regulation onto a V(H)-promoter-Cmu reporter gene, transgenic mice were generated. These mice expressed high serum levels of protein from the transgene. Moreover, high levels of transgene expression were observed in spleen and thymus, while lower expression was found in heart and kidney and no expression was detected in liver and brain. Interestingly, transgene expression was confined to large, activated B cells and peritoneal B cells but not observed in small, resting splenic B cells or activated T cells. However, upon mitogenic stimulation of resting B cells with LPS, high levels of transgene expression was induced. Our data demonstrate that the HS1,2 enhancer can interact with a natural V(H) promoter in a strict temporal fashion and when provided with an appropriate activation signal, this V(H) promoter/enhancer construct can induce transgene expression in resting B, but not T lineage cells. Our data are compatible with a model whereby the regulation of IgH gene expression may be subject to regulation by distinct subsets of cis-regulatory elements acting at different stages of B lymphocyte development. Thus, Ig gene expression may be regulated via an interaction between the V(H) promoter and 3' enhancer elements (here typified by the HS1,2 enhancer) in terminally differentiated B lineage cells.