AbstractThe frequency of B cells specific for 2,4‐dinitrophenyl (DNP), 2,4,6‐trinitrophenyl (TNP) and fluorescein (Fl) in spleen cell suspensions obtained from neonatal, conventionally‐reared, nonimmune adult, and germ‐free adult BALB/c mice was determined by an in vitro splenic focus technique. Per 106 injected adult spleen cells, the frequency of primary B cells stimulated by 10−6M DNP, TNP, or F1 determinant concentration on Limulus polyphemus hemocyanin was 3.5, 3.0, and 2.0, respectively. These frequencies were relatively invariant, even in germ‐free donors. The frequency of DNP and TNP‐specific B cells in neonatal spleens was also relatively invariant and approached adult frequencies early in development. In contrast, variation was observed in the frequency of neonatal precursor cells responding to F1, and spleen cell suspensions from 3‐day old neonates had 5–6‐fold fewer F1‐specific B cells than DNP or TNP‐specific B cells. Since the experimental conditions employed maximized B cell responsiveness, and since the generation of primary B cells appears to be independent of antigenic stimulation, this observed disparity in the rate of appearance of specific B cells is most likely a reflection of the mechanism responsible for generating B cell diversity.