Direct macrophage cytotoxicity against islet cells was examined in a morphological and biochemical study using mouse pancreatic islet cell monolayers cultured in the presence of macrophages. Secretory responsiveness of CBA/J beta cells was tested after 4 days of coculture with syngeneic or allogeneic (C57BL/6J) peritoneal macrophages. Although basal secretion of insulin in response to 5.5 mM glucose was not affected, stimulated insulin secretion in response to 16.5 mM glucose and 5 mM theophylline was reduced by as much as 70% in the presence of syngeneic or allogeneic macrophages. No such effect on stimulated insulin release was observed from cultured islets incubated in the presence of macrophage-conditioned medium. After refeeding the cultures at 4 days, zones of islet cell lysis began to appear wherever macrophages came into contact with islet cells. This macrophage-mediated killing was observed regardless of the source of the macrophages (e.g., intraislet "resident" macrophages, thioglycollate-stimulated peritoneal exudate macrophages, bone-marrow-derived macrophages, and splenic macrophages). The killing was seen with both syngeneic and allogeneic macrophages. Macrophages adjacent to islet cells extended filopodia into the monolayers, and those that adhered to the top of the islet cells formed lytic plaques. This study, by providing direct evidence of macrophage cell-mediated killing of islet cells, suggests the value of eliminating resident macrophages prior to islet transplantation into syngeneic or allogeneic hosts.