B cells expressing two different Ig kappa L chains (allotype included) have been occasionally observed. To determine frequency and function of these cells, we have analyzed gene-targeted mice that carry a human and a mouse Igk C region genes. Using different methodologies, we found that cells expressing two distinct kappa-chains were 1.4-3% of all B cells and that they were present in the follicular, marginal zone, and B1 mature B cell subsets. When stimulated in vitro with anti-IgM, dual kappa surface-positive cells underwent activation that manifested with cell proliferation and/or up-regulation of activation markers and similar to single kappa-expressing B cells. Yet, when activated by divalent reagents that bound only one of the two kappa-chains, dual kappa B cells responded suboptimally in vitro, most likely because of reduced Ag receptor cross-linking. Nonetheless, dual kappa B cells participated in a SRBC-specific immune response in vivo. Finally, we found that Ig allotype-included B cells that coexpress autoreactive and nonautoreactive Ag receptors were also capable of in vitro responses following BCR aggregation. In summary, our studies demonstrate that Ig kappa allotype-included B cells are present in the mouse mature B cell population and are responsive to BCR stimulation both in vitro and in vivo. Moreover, because in vitro activation in response to anti-IgM was also observed in cells coexpressing autoreactive and nonautoreactive Abs, our studies suggest a potential role of allotype-included B cells in both physiological and pathological immune responses.