Spleen cells from transgenic mice, whose rearranged Ig receptors reflect the repertoires of B1 (CD5+ and "sister" B cells) or normal B2 cells, were examined for their ability to be rendered unresponsive. By using an anti-Ig tolerance protocol that is independent of receptor specificity, we previously reported that peritoneal B cells, containing primarily CD5+ and "sister" B cells, were not susceptible to unresponsiveness. Herein, we show that splenic B cells from two separate transgenic mouse lines, each expressing a B1-type receptor, are resistant to tolerance induction in vitro. In contrast, splenic B cells from two other transgenic mouse lines with a large representation of conventional B cells were sensitive to anti-lg-mediated unresponsiveness. This difference does not reside in the surface Ig density, cell cycle, or activation stage of these cells, but is reflected in the initial calcium mobilization and tyrosine phosphorylation after surface Ig cross-linking. Therefore, these results support the hypothesis that the antigenic specificity of B cell receptors may drive cells toward the B1 subset, as suggested by Cong et al. (Cong, Y-Z., E. Rabin, and H. H. Wortis. 1991. Int. Immunol. 3:467-476), and that B1 cell characteristics confer the ability of B cells to withstand in vitro tolerance induction, irrespective of their anatomical location. The possibility that this results from previous antigenic experience is discussed.