The spontaneous proliferation of peripheral blood mononuclear cells (PBMCs) is a hallmark of the human T-lymphotropic virus (HTLV) type-1. Cell proliferation is usually measured using a [(3)H]thymidine incorporation assay. This study aims to quantify the spontaneous proliferation of PBMCs using flow cytometry. PBMCs were cultured for 24 to 120 hours in the presence of 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE). For comparison, PBMCs were also cultured with [(3)H]thymidine. The cutoff values for spontaneous proliferation were >0.06 for the division index and >5.8% for the percentage of divided cells. Sixty-two percent of HTLV-1-infected individuals presented spontaneous proliferation of PBMCs, which was detected in the first 24 hours. Moreover, proliferation was detected in CD4(+) and CD8(+) T-lymphocyte subsets. A positive correlation was found between the division index and [(3)H]thymidine incorporation. This method may prove useful to better understand the phenomenon of spontaneous proliferation of PBMC of patients infected with HTLV-1.