Human monocytes purified by adherence and prolonged in vitro monolayer culture were activated by supernatants of autologous lymphocytes stimulated with live bacillus Calmette-Guérin or killed Corynebacterium parvum. Activated monocytes expressed increased ability to lyse K-562 cells prelabelled with methyl-3H-thymidine in a 48 h assay. Activation could be detected at a 1:64 dilution of lymphokine supernatants. Target cell killing by activated monocytes was strongly influenced by the density of the monocyte monolayer. Monocytes at an intermediate stage of in vitro differentiation seemed to be most response to lymphokine activation. Morphological studies indicated that a considerable number of K-562 cells attach firmly to monocytes. The monocyte-mediated lysis of K-562 seems to be extracellular, as phagocytosis was not observed. Freshly isolated adherent blood mononuclear cells (greater than 90% monocytes) were strongly cytostatic to K-562 cells, as determined by inhibition of methyl-3H-thymidine uptake. Considerable cytolytic activity was also found with freshly isolated adherent cells, strong enhancement being produced by performing the assay in the presence of newborn calf serum, as opposed to human AB serum.