Murine phosphatidyl choline (PtC)-specific B cells in normal mice belong exclusively to the B-1 subset. Analysis of anti-PtC (VH12 and VH12/Vκ4) transgenic(Tg) mice indicates that exclusion from B-0 (also known as B-2) occurs after immunoglobulin gene rearrangement. This predicts that PtC-specific B-0 cells are generated, but subsequently eliminated by either apoptosis or differentiation to B-1. To investigate the mechanism of exclusion, PtC-specific B cell differentiation was examined in mice expressing the X-linked immunodeficiency (xid) mutation. xid mice lack functional Bruton's tyrosine kinase (Btk), a component of the B cell receptor signal transduction pathway, and are deficient in B-1 cell development. We find in C57BL/6.xid mice that VH12 pre-BII cell selection is normal and that PtC-specific B cells undergo modest clonal expansion. However, the majority of splenic PtC-specific B cells in anti-PtC Tg/xid mice are B-0, rather than B-1 as in their non-xid counterparts. These data indicate that PtC-specific B-0 cell generation precedes segregation as predicted, and that Btk function is required for efficient segregation to B-1. Since xid mice exhibit defective B cell differentiation, not programmed cell death, these data are most consistent with an inability of PtC-specific B-0 cells to convert to B-1 and a single B cell lineage.