With appropriate culture conditions, numbers of proliferating foci are a linear function of the number of B cells placed in mitogen-stimulated semisolid cultures, and colony formation is not obviously suppressed or enhanced by T cells. This made it possible to determine the fraction of Ig⁺ B cells in different tissues and stages of development which was capable of colony formation. No evidence was obtained that these functional cells were enriched among either mature or immature B cell populations. Cytolysis with A.TH anti-A.TL serum and C before culture was used to study expression of Ia antigens on colony-forming cells. By this criterion, multipotential stem cells, granulocyte-macrophage progenitors, and fetal colony-forming B cells were Ia negative, whereas all clonable B cells in adult tissues were Ia positive. Colony formation by adult lymph node cells was inhibited by an average of 79% by the addition of rabbit anti-mouse IgD to the cultures, and extremely small quantities of antiserum were required. Adult spleen and bone marrow contained a higher percentage of antiserum-resistant and presumably δ^- B cells, and clonable B cells in fetal tissues were not significantly inhibited. An additional subpopulation of late appearing B cells whose proliferation was resistant to anti-Ig antibodies was identified with a particular set of culture conditions. Colony-forming cells were also heterogeneous in their resistance to preincubation with anti-immunoglobulin antibodies. Under conditions that did not significantly affect adult splenic B cells, clonable cells in fetal and neonatal tissues were substantially inactivated. It appears that colony-forming B cells comprise an equivalent proportion of the B cells in different sites and stages of development and include cells that differ in maturity by the criteria employed here. However, they do not seem to be representative of B cells as a whole, and when considered in relationship to the defect in CBA/N mice, the possibility emerges that the cloning technique preferentially detects a lineage of functionally specialized B cells.