In 70 B-CLL patients, deletion or translocation, at or near the retinoblastoma (Rb) site, was detected in 20 by cytogenetic analysis. Purified B cell clones from 13 of these B-CLL patients were isolated and studied for Rb gene status, Rb mRNA and the Rb protein product. Southern blot analysis of the Rb site detected internal deletions (N = 1) or a single allele loss (N = 2) in five patients. Northern blots detected reduced Rb mRNA in four patients. Immunoblot of whole cell lysate revealed reduced levels of unphosphorylated Rb protein in six CLL patients. No CLL B cell clone contained phosphorylated Rb species. These molecular studies have confirmed the cytogenetic alteration of 13q12-14 sites in B-CLL cells. In addition, cytogenetic and molecular biologic analysis suggest heterogeneity in the B cell clone for Rb gene abnormality. B-CLL patients with abnormalities in both cytogenetic and Rb DNA/RNA analysis will have a dominance of B cells with an Rb abnormality (N = 5). In patients whose Rb defective CLL cells constitute only a minor subpopulation of the total B cell clone, only cytogenetic defects would likely be detected (N = 7).