The activation signals leading to proliferation of leukemic CD3+ large granular lymphocytes (LGL) are incompletely understood. In this study, the role of recombinant human interleukin-12 (rhIL-12) alone or in combination with other activation signals was studied in vitro. Anti-CD3 monoclonal antibody (MoAb) alone caused marked stimulation of peripheral blood mononuclear cells (PBMC) from three CD3+ LGL leukemic patients, whereas rhIL-12 alone had less effect as measured in a [3H]thymidine incorporation assay. The combination signals of anti-CD3 MoAb and rhIL-12 produced a proliferative response greater than anti-CD3 MoAb alone or rhIL-12 alone. Leukemic LGL, purified by CD8+ affinity chromatography, showed similar proliferative responses as PBMC from LGL leukemic patients, suggesting that the observed effect was indeed due to direct stimulation of leukemic LGL. Radiolabeled IL-12 binding studies demonstrated that anti-CD3 MoAb upregulated the number of IL-12 receptors per cell on PBMC from these patients. Neutralizing antibody to rhIL-12 partially blocked the proliferative response to anti-CD3 MoAb suggesting involvement of IL-12 in the pathway of activation of leukemic LGL via stimulation of the T cell receptor (TCR) (mimicking activation by antigen). These results show that IL-12 acts as a costimulatory cytokine for proliferation of leukemic LGL activated through the TCR in vitro.