Abstract Spleen cells from unprimed adult, 7-day-old germ-free, and 3-day-old neonatal mice were separated by 1 × G sedimentation velocity, and the resulting fractions were tested in an in vitro splenic focus assay for NIP-specific precursor frequencies, kinetics of the response, and the class of antibody produced by the NIP-specific clones. The results of this study indicate that the splenic focus assay, contrary to other functional B cell assays, is able to detect the responses of small, medium, and large lymphocytes from both adult and neonatal B cell sources. Evidence is presented for a time-dependent appearance of hapten-specific clones in some of the B cell fractions. Most striking is the very large cell subset sedimenting at an s value >5.5 in the adult and >6.1 in the neonate which shows a progressive increase in the frequency of hapten-specific precursors. This would suggest the presence of a less mature B cell. An analysis of the class of antibody produced by the various sizes of B lymphocytes from both adults and neonates indicated that the majority of B cells in the smallest and largest cell fractions tested produce IgM only, whereas the majority of B cells in the medium-sized lymphocyte fraction produced IgM or IgM plus IgG classes. In addition, the cycling status of adult and neonatal lymphocytes in the various size distributions was examined. It was found that the smallest lymphocyte fraction contained no detectable lymphocytes in rapid cell cycle. The largest lymphocyte fraction, on the other hand, was enriched for cycling cells over unfractionated controls, and in the neonatal fraction the majority of lymphocytes were in rapid cell cycle. The splenic focus assay is presumably capable of detecting a wide range of B cell subsets as defined by the criteria above including less mature B cells and is therefore a very useful cloning assay for studying B cell development.