Persistent polyclonal B-cell lymphocytosis (PPBL) is an extremely rare disorder, which occurs almost exclusively in smoking women and is characterized by a lymphocytosis with circulating binucleated lymphocytes. We analyzed 25 PPBL patients with respect to immunophenotype and by adaptive cluster analysis system (ACAS). Furthermore, HLA type, presence of Epstein-Barr virus (EBV) DNA in B cells, and clinical data were evaluated. Overall, the median percentages of B cells in PPBL patients with expression of CD5(dim), CD23(dim), CD25, CD27, and FMC7 were 21%, 38%, 16%, 74%, and 93%. Compared to normal controls, ACAS revealed a subset of nucleic-acid-rich lymphocytes located between the regular lymphocyte and regular monocyte region. Sixteen (64%) of 25 patients carried a HLA DR7 phenotype. Quantitative real-time polymerase chain reaction analysis did not detect relevant amounts of EBV DNA in circulating B cells of any patient. During a median follow-up of 5 years, a single patient developed lymphoplasmacytic lymphoma. The abnormal morphology and frequent, albeit dim, expression of CD5 and CD23 in PPBL may result in erratic diagnostic assignment of this benign disorder. However, incorporation of immunophenotyping and ACAS into the diagnostic algorithm allows recognition of PPBL in routine analysis and its differentiation from malignant B cell lymphoproliferative diseases. We found that an infection of a significant percentage of PPBL cells by EBV is unlikely. The observation of malignant lymphoma in a single patient implicates that evolution into a clonal malignant transformation may occasionally occur in PPBL.