Fc gamma receptor I-positive (CD64+) and Fc gamma receptor I-negative (CD64-) monocytes were prepared from highly purified (elutriation-derived) human monocytes by cytofluorograph cell sorting, and a phenotypical and functional dissociation of the isolated CD64+ and CD64- monocyte subsets is demonstrated. Surface analyses revealed that the surface antigen pattern of CD64+ monocytes corresponds to the phenotype of typical unseparated monocytes. In contrast, CD64- monocytes are characterized by high expression of major histocompatibility complex (MHC) class I antigens (HLA-A, -B, -C) and MHC class II antigens (HLA-DR, -DP, -DQ), and low expression of the monocyte-specific marker CD14 which is found on nearly all CD64+ monocytes. However, 75% of the CD64- cells were found to be esterase-positive, and 85% were positive for the the CD64- cells were found to be esterase-positive, and 85% were positive for the monocyte/macrophage-specific intracellular antigen CD68. Furthermore, CD64- monocytes show significantly higher expression of CD45RA and Fc gamma receptor III (CD16) than CD64+ monocytes, but lack the natural killer cell markers CD56 and CD57. Functional studies showed that cells of the minor CD64- monocyte subset have a higher accessory cell capacity in antigen-driven T cell activation than CD64+ monocytes. CD64- monocytes pretreated with PPD (purified protein derivative of tuberculin) induced up to tentimes more interferon-gamma and also higher proliferation in responding autologous T cells than PPD-pretreated CD64+ monocytes. Similar results were obtained for T cells in mixed leukocyte reaction. Interferon-gamma release and proliferation of allogeneic lymphocytes were consistently higher in the presence of irradiated CD64- monocytes than of irradiated CD64+ monocytes. Furthermore, when CD64- and CD64+ monocytes were stimulated with Newcastle disease virus, we measured an up to 67-fold higher interferon-alpha release from CD64- than from CD64+ monocytes, indicating a higher anti-viral capacity of this subset. CD64- monocytes showed lower activity in the phagocytosis of unopsonized particles and also lower zymosan- or latex-induced chemiluminescence than CD64+ monocytes. These findings indicate that CD64- monocytes, although comprising only less than 10% of all peripheral blood monocytes, represent a monocyte subpopulation efficiently interacting in vitro with T cells and, additionally, are the major source of interferon-alpha.