These studies were undertaken to examine the phenotype of the antigen-presenting cells (APC) circulating in human peripheral blood. Cells adherent to glass were found to be efficient APC, restoring antigen-induced 3H-thymidine incorporation to T4-positive T cells that had been rigorously depleted of contaminating APC. In order to identify the APC within the glass-adherent cells (AC), these cells were stained with a number of monocyte-specific monoclonal antibodies (Mo-Mab) including 3C10, 63D3, and 61D3, and the Mo-Mab-positive and -negative cells were separated with the fluorescence-activated cell sorter. This method of preparation yielded Mo-Mab(+) AC populations that were more than 98% positive for the relevant Mab when reanalyzed with the fluorescence-activated cell sorter. Less than 1% of the Mo-Mab(-) AC populations were positive when reanalyzed with the Mab used for the separation. However, each Mo-Mab(-) AC population was contaminated with variable numbers (4-60%) of Mo as detected by morphologic criteria, histochemical analysis for esterase activity, or staining with a different Mo-Mab. Both Mo-Mab(+) and (-) AC populations were found to be similarly effective APC, with as few as 500 cells/well supporting responses to streptokinase-streptodornase, tetanus toxoid, and Candida albicans antigen. In the absence of antigen, only 3C10(-), 63D3(-), or 61D3(-) AC consistently stimulated 3H-thymidine incorporation of autologous T4 cells; large numbers (greater than 5 X 10(3)/well) of APC were necessary to induce this response. These results support the conclusion that cells identified by Mo-specific Mab are capable of functioning as APC, inducing 3H-thymidine incorporation in response to exogenous antigens. However, Mo-Mab(+) AC are not unique in this activity since Mo-Mab(-) AC also appeared to be able to present antigen. These Mo-Mab(-) AC appear to contain the majority of cells inducing autologous T4-cell reactivity.