We previously observed that pseudorabies virus (PRV)-induced, cell-mediated cytolysis in pigs includes killing by natural killer (NK) cells. We also observed that IL-2 stimulation in vitro of naive PBMC expands porcine NK cells. The purpose of this study was to compare the phenotypes of the cytolytic subsets stimulated in vitro by PRV and by IL-2. PBMC were isolated from blood of PRV-immune and naive pigs and stimulated in vitro with PRV or IL-2. After 6 days, the frequency of various lymphocyte subsets in these cultured PBMC was determined by flow cytometry: the cells were separated with a magnet-activated cell sorter and the cytolytic activity of the separated populations was determined. When lymphocytes were separated and analysed with FACScan, the following lymphocyte subsets were discriminated: CD6(+) CD8(bright+) CD4(-) (CTL phenotype), CD6(+) CD8(dull+) CD4(+) (the fraction containing memory T helper cells), CD6(+) CD8(-) CD4(+) (T helper cell phenotype), CD6(-) CD8(dull+) CD4(-) gammadelta-T(+) ( gammadelta-T cell phenotype), CD6(-) CD8(dull+) CD4(-) gammadelta-T(-) (NK phenotype) and CD6(-) CD8(-) CD4(-) gammadelta-T(-) or gammadelta-T(+). Flow cytometry analysis demonstrated that PRV stimulation of immune PBMC resulted in the occurrence of more CD6(+) CD8(+) and CD4(+) CD8(+) and fewer CD6(-) CD8(+) and gammadelta-T(+) CD8(+) lymphocytes than IL-2 stimulation of naive PBMC (P<0.05). It was demonstrated further that killing by PRV-stimulated PBMC was mediated mainly by CD6(+) CD8(+) T lymphocytes. Killing by IL-2-stimulated PBMC was mediated mainly by CD6(-) CD8(+) T lymphocytes. These results demonstrate that both natural killing and killing by classical PRV-specific CTL were detected in PRV-immune pigs, whereas IL-2 stimulation of PBMC isolated from naive pigs mainly induced natural killing.