Chronic lymphocytic leukemia of B-cell origin (B-CLL) is a disease with a variable clinical course, despite the fact that the neoplastic cells in this disorder are homogeneous with respect to morphology, immunophenotype, and cell cycle stage. To further investigate the heterogeneity observed in the clinical behavior of B-CLL, we determined the phenotype and growth requirements of clonogenic cells from 28 patients with B-CLL from low-, intermediate-, and high-risk groups as defined by the Rai staging system. Using methyl-cellulose as a semi-solid media with feeder cells and/or growth factors, colonies were observed with one or more of the culture conditions tested in 25 of 28 CLLs. Phenotypic analysis of colonies demonstrated that the clonogenic cells uniformly expressed la, CD19, CD20, CD5, and the identical light chain as the original CLL cell cultured. However, heterogeneity was observed in clonogenic B-CLL cell growth among the three different CLL risk groups. Clonogenic cells from patients with low-risk CLL required either irradiated unstimulated T cells, with or without conditioned media (CM) or irradiated activated T cells alone for colony formation. Both the number of colonies (227 +/- 15) as well as the number of cells per colony (220 +/- 82) were large, with a mean cloning efficiency of 0.39%. In contrast, clonogenic cells from patients with intermediate- and high-risk CLL required the combination of both irradiated activated T cells and CM. As compared with the low-risk CLLs, both the number and size of the colonies formed by the intermediate- (74 +/- 17, 70 +/- 39) and high- (83 +/- 28, 40 +/- 14) risk groups were significantly lower (P less than .0001). Similarly, the mean cloning efficiency was significantly reduced to 0.15% and 0.14%, respectively. None of the recombinant cytokines (interleukin 1 [IL-1] to IL-7, tumor necrosis factor, alpha and gamma-interferon, B-cell growth factor, and granulocyte macrophage colony-stimulating factor) alone or in combination with each other could entirely replace the stimulatory effect of the activated T cells. These data suggest that clinical progression of B-CLL is associated with a loss of clonogenic potential in the circulating pool of neoplastic cells, which require as yet undefined factors provided by activated T cells and CM.