We have previously used human Epstein Barr virus (EBV)-transformed B lymphoblastoid cell lines for the identification and purification of a novel cytokine, natural killer cell stimulatory factor (NKSF/IL-12), that has pleiotropic effects on human lymphocytes. B cell lines are also routinely employed as feeder cells for the culture of T and natural killer (NK) cells. In this report we describe the ability of two NKSF/IL-12 producing B cell lines (RPMI-8866 and Cess) and two nonproducing lines (Raji and Daudi) to stimulate the proliferation of T and NK cells in 8-day PBL cultures. We demonstrate, using an anti-NKSF/IL-12 neutralizing monoclonal antibody, that the endogenous production of NKSF/IL-12 in these cultures can significantly enhance the proliferation and cytotoxic activity of T and NK cells. We also report that the addition of exogenous rNKSF/IL-12 can greatly increase the number of T and NK cells obtained from the cultures following stimulation by the B cell lines. Aside from the possible practical applications, the enhanced proliferation of T and NK cells consistently observed in the presence of endogenously produced NKSF/IL-12 or exogenously added rNKSF/IL-12 in this system may further our understanding of the role of this cytokine during an in vivo immune response.