The emergence of functional B cells was monitored in irradiated or unirradiated CBA/N recipients of either adult bone marrow or fetal liver from CBA/HT6T6 donors. The cells that are primarily responsible for the generation of B lymphocytes, at least during the first 6 wk, are rapidly sedimenting (4.5-6 mm/h), lack surface immunoglobulin, and are found in both the adult bone marrow and the fetal liver from day 12 onward. These pre-B cells are distinct from the colony-forming unit spleen (CFU-s) as demonstrated by the following criteria: (a) absence from yolk sac (19), (b) lack of correlation between CFU-s number and the ability to generate B cells in fetal liver populations of different ages of gestation, and (c) hybridoma antibodies that significantly inhibited B cell reconstitution but have no effect on CFU-s numbers. The antigen detected by this antiserum is present on both the fetal liver and bone marrow B cell progenitor, although its expression is not restricted to the B lineage. The pre-B cells that we monitor are not homogeneous, however, as both physical and functional differences are found. These observations reinforce our thesis that committed progenitor cells for the humoral immune system are formed early in development and thereafter constitute the major precursor pool for the generation of B lymphocytes.