The incorporation of radioactive nucleotides into newly synthesized DNA has been established as a standard method for the detection of proliferation in eucaryotic cells. Unfortunately the use of this method makes it harder to obtain information on the phenotype of proliferating cells in mixed cell populations. For this reason we established a flow-cytometric approach employing a monoclonal antibody specific for murine as well as human proliferating cell nuclear antigen (PCNA) and a double labeling technique for detection of cell membrane-expressed phenotypic markers. The efficiency of this immunostaining procedure was confirmed by simultaneous and highly specific detection of PCNA in nuclear structures as well as cell membrane-expressed antigens using cytological techniques. In vitro experiments with mitogen- and alloantigen-stimulated murine lymph node cells (LNC) and human peripheral blood mononuclear leukocytes (PBML) revealed a good correlation of total [3H]thymidine incorporation into DNA and expression of PCNA. For the analysis of proliferating cells activated in vivo the method was employed to evaluate the local lymph node assay which assesses the allergenicity of small chemicals. LNC prepared from the cervical lymph nodes of mice treated on 4 consecutive days with sensitizing concentrations of the contact allergens oxazolone, TNCB and DNFB as well as the irritants benzoic acid and SLS in comparison to the solvent control showed a dramatic increase in the total amount of proliferating cells for contact allergen-treated animals in comparison to the solvent control and irritant-treated mice. In addition a detailed phenotyping of the proliferating cell populations was possible. This approach offers an easy to perform, non-radioactive method for the assessment of proliferation of murine as well as human leukocytes in vitro and especially in vivo and will be of great advantage for situations where the phenotype of proliferating cellular subsets in heterogeneous populations is of interest.