Inactivation of the retinoblastoma tumor-suppressor gene (RB-1) has been associated with tumorigenicity in various human malignancies. In chronic lymphoid leukemias of B-cell origin (B-CLL) an involvement of RB-1 has been suggested based on cytogenetic data. We examined RB-1 and its chromosomal locus 13q14 in 35 cases of B-CLL by dual-color in situ hybridization to interphase nuclei and by G-banding analysis of metaphase chromosomes. In one patient (pt) a monosomy 13, and in three other pts deletions involving or encompassing band 13q14 were detected by conventional cytogenetic analysis. In contrast, in situ hybridization to interphase nuclei showed a monoallelic RB-1 deletion in 11 cases (31%). One pt showed a translocation with the breakpoint in 13q1?4 on G-banding, but on in situ hybridization analysis the RB-1 signals were not affected. Our data show that RB-1 deletions can be diagnosed accurately by in situ hybridization on the one-cell level. The frequency of RB-1 deletions detected in this study is significantly higher than previously assumed in B-CLL, and seems to be in the same range as in retinoblastoma.