Adherent monolayers of freshly isolated human peripheral blood mononuclear cells consist mostly (greater than 90%) of monocytes. The initially adherent nonmonocytic cells detach during the first day of in vitro culture. Concomitant with cell detachment the cytotoxicity mediated by adherent monolayers is reduced. This is in part due to medium exchange with removal of initially adherent cells from the monolayers, cells that are potent mediators of cytostasis and cytolysis. The temporarily adherent cells consist of 10% monocytes, 35% T lymphocytes, 10% B lymphocytes, and 45% non-T, non-B lymphocytes. In addition, 45% of these cells stain positively with the monoclonal antibody OKT 10. The reduction in monolayer-mediated cytoxicity from day 0 to day 1 of in vitro culture is less pronounced if the temporarily adherent cells are kept within the culture during the cytotoxic assay time. When the temporarily adherent cells are returned to adherent monocyte monolayers an additive effect on cytolysis is demonstrated with nonactivated, lymphokine-activated and alpha-interferon-activated effector cells, and in antibody-dependent cell-mediated cytotoxicity. The heterogenous population of temporarily adherent cells in freshly isolated monolayers of human mononuclear blood cells seems to be responsible for some of the cytotoxic effects regularly ascribed to freshly isolated human blood monocytes.