Abnormalities of chromosome 14 involving band q32.33 are among the most commonly observed cytogenetic alterations in B-cell malignancies. To assess the incidence and pathogenetic implications of 14q32.33 translocation in chronic B-cell leukemia/lymphomas, we performed fluorescence in situ hybridization (FISH) analysis with variable region (V(H)) and gamma constant region (Cgamma) gene probes in 37 patients with these disorders. Chromosome 14q32.33 translocation was detected in 2 of 18 patients with chronic lymphocytic leukemia (CLL), 1 of 2 with CLL of mixed cell types (CLL/PL), 1 of 2 with pro-lymphocytic leukemia (PLL), 5 of 6 with leukemic mantle-cell lymphoma (MCL), 2 of 7 with splenic B-cell leukemia/lymphoma of possible marginal zone origin (SBLL) and 2 with leukemic follicular lymphoma (FL). To further characterize 14q32.33 translocations in these patients, we developed a new procedure using double-color FISH with PRAD1, BCL2, V(H) and Cgamma gene probes. Chromosome t(11;14) was detected in 1 patient with CLL/PL, 1 with PLL and 5 with MCL. Chromosome t(14;18) was detected in 2 patients with FL. In a PLL patient with t(11;14), the cosmid CPP29 containing the PRAD1 gene and its 5'-flanking region split and co-localized with both Cgamma and V(H) gene probes, thus spanning the breakpoint. In CLL and SBLL patients, donor chromosomes were other than chromosomes 2, 11, 18 and 19, suggesting the involvement of a novel oncogene(s) in the pathogenesis of these diseases. Interphase FISH rapidly detected 14q32.33 translocation, t(11;14) and t(14;18) in B-cell malignancies with low mitotic activity at the single-cell level, facilitating the correlation of the molecular features of these translocations with clinical characteristics.