During their development B lymphocytes pass through a maturational stage in which encounter with Ag leads to tolerance rather than activation. At least four mechanisms for achieving B cell tolerance have been reported: deletion, anergy, receptor editing, and competition for follicular niches. Although turnover rates for immature B cells in the adult mouse bone marrow and several transgenic model systems suggest that a major process contributing to negative selection of B cells is deletion, a detailed study of the negative effect of Ag-receptor engagement on primary, immature B cell survival has never been undertaken. We have utilized an in vitro culture system to determine whether cross-linking sIgM on tolerance-susceptible sIgM+IgD- B cells results in deletion by apoptosis. In contrast to the effect of sIgM cross-linking on mature splenic B cells, treatment of immature, bone marrow-derived B cells results in significant levels of apoptotic death. Ag receptor-mediated apoptosis is detectable by 14 h after sIgM engagement. Moreover, IL-4 and cycloheximide, which have previously been shown to prevent B cell tolerance induction, specifically block the sIgM-induced apoptosis observed in the immature B cells. Similarly, immature B cells from the neonatal spleen are also susceptible to apoptosis after sIgM cross-linking, although they manifest somewhat higher levels of unstimulated apoptosis as compared with bone marrow-derived B cells. These studies are the first detailed demonstration of Ag receptor-mediated apoptosis of primary immature stage B lymphocytes.