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Stimulation of lymphocyte proliferation using mitogens or specific antigens is a method that is used frequently to assess immune responsiveness. While useful, lymphocyte blastogenesis, or [3H]-thymidine incorporation, provides little information regarding the response of specific subsets to the stimulant. Here, we report that the fluorescent cell membrane probe, PKH2, is a useful tool for measuring the proliferation of porcine lymphocyte subpopulations by utilizing multicolor flow cytometry. For this study, mitogen-induced proliferation of porcine peripheral blood mononuclear cells (PBMCs) was measured using [3H]-thymidine incorporation as well as a flow cytometric-based proliferation assay. From the [3H]-thymidine incorporation data alone, it was observed that PBMC stimulated with either concanavalin A (Con A), phytohemagglutinin (PHA) or pokeweed mitogen (PWM) demonstrated greater proliferation on day 3 than on day 5 of culture. Using the PKH dye and flow cytometric analysis, the responsiveness of specific lymphocyte subsets to mitogen stimulation was detected. The predominant subsets of porcine lymphocytes responding to Con A or PHA stimulation were CD4+CD8+, CD4−CD8αhi, CD4−CD8αlo and γδ TCR+ cells. PWM stimulation induced responses by CD4+CD8+, CD4CD8αhi but not by CD4−CD8αlo or γδ TCR+ cells. Con A stimulation resulted in a sustained proliferation of CD8αhi cells over the 5-day period while PHA stimulation resulted in proliferation that peaked within the first 3 days. Little or no proliferative responses were detected within the IgM+ population (e.g. B cells). This is the first study to define the contribution of individual lymphocyte subsets to mitogen-induced proliferation of porcine PBMCs. [Copyright &y& Elsevier]