T wo human monocyte subsets from the peripheral blood of healthy donors have been isolated in greater than 90% purity by countercurrent centrifugal elutration and human serum albumin gradients and their functional capabilities have been assessed. We have demonstrated that one subset ("regular" monocytes, RM) showed intense cytoplasmic peroxidase staining and contained substantial peroxidase activity. In contrast, another subset ("intermediate" monocytes, IM) stained poorly for peroxidase and had low peroxidase activity. By electron microscopic analysis combined with peroxidase localization, it was found that IM had fewer peroxidase-positive granules per cell than did RM. IM coelutriated with some lymphocytes and by cell sizing analysis were shown to be slightly smaller than RM. Functional and cytochemical analysis of these subsets indicated that IM had less activity than RM in assays such as accessory cell function for mitogen-induced T lymphocyte proliferation and antibody-dependent cellular cytotoxicity, and that fewer IM expressed OKM1 antigen and pokeweed mitogen (PWM) receptors on their membranes than did RM. The subset of IM not bearing either the PWM receptor or the OKM1 antigen had very low peroxidase activity. IM also were found to have a greater sensitivity to polyriboinosinic and polyribocytidilic acid (100 micrograms/ml)-induced secretion of interferon. There was no significant difference in the phagocytic capability, the percentage of Fc receptor-positive cells, 5'-nucleotidase activity, DR antigen expression, or the responsiveness to migration inhibitory factor of IM as compared with RM. Furthermore, it was found that the ratio of IM to RM increased after prolonged cytapheresis, which suggests that IM are more mobilizable than RM from the extravascular reservoirs of human monocytes.