Serological techniques were used to examine the process of sequential surface antigen expression on differentiating B cells in vitro. A 4-day culture system is described in which bone marrow lymphocytes from neonatal mice acquire in sequence the ability to express Lyb-2, IgM, Ia and IgD in response to a 3-h induction with E. coli lipopoly-saccharide (LPS). Lyb-2 can be induced on day 1, IgM can be induced after 24 h, Ia after 48 h and IgD only after 96 h in culture. This sequence mimics the order of appearance of B cell surface antigens during ontogeny. When DNA synthesis is blocked from 0.24 h with hydroxyurea (HU), all surface antigens can be induced simultaneously by LPS. Immunoselection of one antigen-bearing population results in the loss of cells bearing other B cell antigens indicating that the surface antigens are induced on the same cells. When both HU and LPS were added to the cultures from the start, IgM appears after 11-14 h, Ia afer 14-15 h and IgD only after 19 h. Induction of antigen was demonstrated by he cytotoxicity assay, quantitative absorption and the protein A sheep red blood cell rosetting assaying. The results obtained show that there is a population of surface IgM-negative precursor B cells in young bone marrow which, when grown in vitro, become sequentially inducible for expression of B surface antigens. Inhibition of DNA synthesis promotes acquisition of the inducible state, and the sequence of antigen expression is correlated with specific time intervals after DNA synthesis has stopped.