Peripheral blood mononuclear cells (PBMC) from normal individuals were studied to identify which cells produce alpha-interferon (IFN-alpha) in response to a virus stimulus. It was found that cells both adherent and nonadherent to plastic formed IFN-alpha after induction by any one of several viruses studied. When nonadherent cells were separated on discontinuous Percoll gradients, only the cells in the less dense Percoll fractions produced IFN, whatever the virus used. By indirect immunofluorescence with monoclonal antibodies to HLA-DR and to Leu 11b, the distribution of the HLA-DR+ cells was shown to resemble most closely that of the IFN-producing population. Elimination of these cells (by complement-mediated lysis with the same antibodies) abrogated the IFN response, but NK cells remained and thus do not produce IFN-alpha. In confirmation, elimination of the Leu 11b+ cells had no effect on the amount of IFN produced. PBMC preparations from patients with chronic lymphocytic leukemia (CLL) appeared incapable of producing IFN-alpha but were shown to contain identifiable IFN-producing cells. The low or absent IFN levels in CLL are probably due to the relative scarcity of IFN-producing cells in their PBMC.