The role of HLA-DR+ cells in NK activity against CMV-infected FS4 foreskin fibroblasts and K562 erythroleukemia cells was examined. When nonadherent PBMC were depleted of either HLA-DR+ or Leu-11b+ cells by treatment with mAbs plus C, NK activity against CMV-FS4 target cells was markedly reduced. In contrast, depletion of HLA-DR+ cells had no effect on NK activity against K562 target cells. When HLA-DR-depleted cells were added to Leu-11b-depleted cells, NK activity against CMV-FS4 was restored. Negative selection experiments indicated that the HLA-DR+ cells contributing to NK activity against CMV-FS4 are not B or T cells, while negative and positive selection experiments excluded a role for monocytes. Experiments in which HLA-DR- and Leu-11b- cells were mixed in varying proportions indicated that NK(CMV-FS4) is mediated by Leu-11b+ cells, while HLA-DR+ cells provide an accessory function. Irradiation (50 GY) abolished the NK effector function of Leu-11b+ cells, but not the accessory function of HLA-DR+ cells. The NK activity against CMV-FS4 of HLA-DR- cells was restored by the addition of rIFN-alpha or of cell-free supernatants generated by coculturing PBMC or Leu-11b- cells with CMV-FS4. The ability of these supernatants to restore NK activity of HLA-DR- cells was completely abrogated by the addition of neutralizing amounts of antibody to IFN-alpha. In related experiments, neutralization of IFN-alpha in NK assays had little or no effect on NK activity against CMV-FS4, suggesting that the accessory function of HLA-DR+ cells might be mediated by alternative mechanisms in addition to the secretion of extracellular IFN-alpha.