The purpose of this study was to characterize the destruction of sensitized erythrocytes by human blood monocytes in vitro. The incubation in vitro of human monocytes with 51Cr-labelled human erythrocytes sensitized with IgG rhesus alloantibodies anti-D (EAIgG anti-D) resulted in release of 51Cr from the erythrocytes (lysis) as well as uptake of 51Cr-labelled erythrocytes by the monocytes (phagocytosis). The lysis of EAIgG anti-D by monocytes was not dependent on phagocytosis, because cytochalasin B, which inhibited phagocytosis of EAIgG, enhanced lysis. In contrast, hydrocortisone and colchicine inhibited lysis, but had no effect on phagocytosis. These agents did not affect binding of EAIgG anti-D to monocytes. The effect of these agents on lysis corresponded to their effect on release of lysosomal enzymes by monocytes. The release of lysosomal enzymes, when induced by EAIgG anti-D, was, likewise, enhanced by cytochalasin B and inhibited by hydrocortisone and colchicine. A significant correlation was found between lysosomal enzyme release and lysis. Together, these results strongly suggest that lysosomal enzymes, released by the monocytes when incubated with anti-D-sensitized erythrocytes, are responsible for the cytotoxic activity of these cells towards sensitized erythrocytes. The action of these enzymes only occurs over a short range, probably at the site of attachment of the erythrocyte, because only erythrocytes that were bound to the monocytes were lysed. The finding of other investigators that removal of monocytes from suspensions of human mononuclear leucocytes results in a strong reduction in the cytotoxic activity of these leucocytes towards sensitized erythrocytes in vitro. was confirmed.