CLL B cells may be induced to form B-cell colonies in vitro. Colonies formed are monoclonal and appear to reflect the circulating malignant B-cell clone in vitro. Using hybridoma-produced monoclonal antibodies (MAB) and an in vitro B-cell colony assay, we have provided a characterization of the antigenic phenotype of the clonogenic CLL B cell. B-cell colony growth in both patients and normals was not altered by prior incubation with either MAB or complement (C') alone. CLL B-cell colony formation was markedly reduced after treatment with T101 and C', while normal colonies were unaffected (8 ± 2 versus 107 ± 10). None of the residual CLL B-cell colonies after T-101 and C' treatment reacted with T-101. However, BA-1 and Ia reactivity were still seen in residual CLL B-cell colonies following T-101 treatment. In contrast, a similar percentage reduction of B-cell colony growth was seen for both normals and CLL patients following treatment with BA-1 (76% versus 81%) and Q5/13 (89% versus 92%). These studies suggest that the CLL progenitor cell is characterized by the phenotype Ia+, BA-1+, T-101+. Better definition of the CLL progenitor cell has potential implications with regards to clinical utilization of MAB in the treatment of CLL.