Twenty-four hours following treatment of rats with the analgesic acetaminophen (1.2 g/kg), we observed an infiltration of mononuclear cells into centrilobular regions of the liver in the absence of necrosis. To determine whether acetaminophen induces the accumulation and activation of mononuclear phagocytes, we compared the morphologic and functional characteristics of macrophages obtained from livers of acetaminophen-treated rats with those of resident macrophages (Kupffer cells) from untreated control animals. Macrophages were isolated from rat livers by combined collagenase/pronase perfusion, selective digestion, and differential centrifugation on a metrizamide gradient. Acetaminophen treatment resulted in a twofold increase in macrophage yields from the liver compared with controls. Macrophages isolated from treated animals were generally larger than resident Kupffer cells, were highly vacuolated, and adhered to culture dishes more rapidly. Liver macrophages from both treated and untreated rats phagocytized sheep red blood cells (sRBC) in a time-dependent manner, reaching a maximum after 60-75 min incubation with sRBCs. However, macrophages from livers of acetaminophen-treated rats phagocytized two to three times more sRBC than did resident Kupffer cells. Using the Boyden chamber technique, both macrophage populations were found to be chemotactic to a number of stimuli including the complement fragment, C5a, and synthetic collagenous peptides related to tissue breakdown products. Levels of migration of macrophages from livers of acetaminophen-treated rats were four to seven times greater than those of resident Kupffer cells. In addition, compared with resident Kupffer cells, macrophages from acetaminophen-treated rats released 30% more superoxide anion in response to the stimulus, 12-O-tetradecanoyl-phorbol-13-acetate. Taken together, these results suggest that acetaminophen treatment of rats leads to the recruitment and activation of macrophages in the liver.