Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare entity of unknown etiology characterized by a polyclonal expansion of B-lymphocytes with typical bilobulated forms, elevated serum IgM, and an additional isochromosome for the long arm of chromosome 3 as the sole change. In the present study, we investigated four cases of PPBL by means of conventional cytogenetic analysis and FISH. In all patients, the polyclonality of the lymphoproliferation was demonstrated by immunophenotypic studies, and PCR analysis failed to demonstrate clonal IGH rearrangements in three evaluated cases. In two patients, in addition to ϩi(3)(q10), banding techniques identified unrelated clones with trisomy 3. FISH studies using a chromosome 3 long arm-specific probe provided evidence that all cases had both ϩi(3)(q10) and ϩ3. To determine more precisely the distribution of the chromosomal abnormalities within the peripheral lymphocyte population, we investigated two of these cases using a technique of simultaneous fluorescence immunophenotyping and interphase cytogenetics (FICTION). We demonstrated that both abnormalities were randomly distributed among the B-lymphocytes, independently of the or light chain isotype and the nuclear aspect. These data lead us to conclude that trisomy 3 represents, in addition to ϩi(3)(q10), another recurrent cytogenetic change in PPBL, suggesting that this lymphoproliferative disorder is associated with an increased frequency of chromosome 3 instability.