Adult mice have two easily recognizable subsets of B cells: the predominant resting population of the spleen, called B-2, and those called B-l, which predominate in coelomic cavities and can express CDS. Some antibody specificities appear to be unique to the B-1 population. Cells expressing antibody specific for phosphatidyl choline (PtC) are the most frequent, comprising 2-10% of peritoneal B cells in normal mice. To understand the basis for the segregation of the anti-PtC specificity to this population, we have produced transgenic (Tg) mice expressing the rearranged VH12 and VKD genes of a PtC-specific B-1 cell lymphoma. We find that VH12-Tg and VH12/VKD double-Tg mice develop very high numbers of PtC-specific peritoneal and splenic B cells. These cells have the characteristics of B-1 cells; most are CD5+, and are all IgM -, B2201- and CD23-. In the peritoneum these cells are also CD11b+. In addition, adult mice have many splenic B cells (up to one third of Tg+ cells) that express the V.12 Tg but do not bind PtC, presumably because they express a V gene other than Vk4. These cells appear to be B-2 cells; they are CD23+ CD11b−, IgMlo B220hi and CD5−. Thus, mice given either the VH12 Tg alone or together with the VK4 Tg develop a large population of PtC-specific B cells which belong exclusively to the B-1 population. Since B-2 cells can express the V.12 and VK4 gene separately, we interpret these data to indicate that the events leading to the segregation of PtC-specific B cells to the B-1 population in normal mice are initiated after Ig gene rearrangement and expression. These data are discussed with regard to hypotheses of the origin of B-1 cells. We also find that VH12-Tg mice have a marked decrease in the generation of Tg-expressing B cells in adult bone marrow, but not newborn liver. We speculate that this may be related to positive selection of VH12-expressing B cells during differentiation.