Human peripheral blood mononuclear leukocytes cocultured with WISH human amnion cells or K562 tumor cells rapidly produce interferon-alpha (IFN). In the present report we characterize the IFN-producing cells (IPC) in this system by cell separation procedures, including panning with different monoclonal antibodies. Two types of cells which were responsible for the IFN production could be identified. The first IPC was classified as monocyte/macrophage because it was present in plastic-adherent cells and apparently carried the OKM1 antigenic marker. T and B lymphocytes were not involved in the IFN production. The second type of IPC was nylon wool-nonadherent, sheep erythrocyte rosette-negative, at least partly Fc receptor-negative and resided in light Percoll density gradient fractions. The natural killer activity and IFN-producing capacity was studied in such cells fractionated by the panning technique, utilizing HNK-1, OKT10 and OKM1 antibodies. When WISH and K562 were used as IFN inducers, HNK-1+ and OKT10+ cells with lytic activity against K562 produced little or no IFN. The IPC were instead confined to HNK-1- and OKT10- cells. With cells fractionated with respect to the OKM1 antigen, natural killer activity against K562 and WISH cells and IFN production stimulated by K562 cells resided in the OKM1+ cells. In marked contrast, cells producing IFN in response to WISH cells were OKM1-. The results therefore demonstrate a dissociation between natural killer cells against tumor cells and IPC stimulated by the same targets, suggesting that for at least certain targets/inducers they may represent distinct entities.