The regulation of phenotype and function of human liver infiltrating lymphocytes (LIL) by in vitro culture with IL-2, IL-12 and anti-CD3 monoclonal antibodies (mAb) was investigated. The CD3+ LIL which express 50% less CD3 molecules per cell than peripheral blood T lymphocytes, exhibited a 6-fold reduction in proliferation when stimulated through the CD3 complex by anti-CD3 mAb. LIL freshly isolated or cultured in medium did not suppress MLR response, nor were they cytotoxic. However, treatment of the LIL cells with IL-2, IL-12 and anti-CD3 induced these cells to suppress autologous responding cells in MLR (ca. 70%) and to kill autologous or allogeneic cells. Low level cytotoxicity could be induced by cytokines IL-2, IL-12 or anti-CD3 alone. However, the development of optimum MLR suppression and cytotoxicity induction was dependent upon stimulation of the LIL cells through the CD3 complex. The co-expression of CD3 and CD56 on LIL was also up-regulated by anti-CD3 stimulation in the combination of IL-2 and IL-12. Most of the CD3+/CD56+ cells, also expressed CD8. After the magnetic bead separation procedure, the cytotoxic activity was found mainly in the CD3+/CD56+/CD8+ population. These results suggest that CD3+/CD56+/CD8+ cells can be expanded by stimulation through the TCR/CD3 complex in the presence of IL-2 and IL-12, which results in the suppression of autologous responding cells by a cytotoxic mechanism. The proliferative response of the CD3+/CD56+/CD8+ population was enhanced by the induction of CD1 molecules on the stimulating cells, and anti-CD1 mAb were able to block the response in a dose-dependent manner. The CD3+/CD56+/CD8+ cells were examined for cytokine production by flow cytometry. Cytokines IL-4, TNF-alpha, and IFN-gamma were produced by 91.7%, 29.2%, and 27.4% of the cells, respectively.