Mouse macrophages derived either from mildly inflamed peritoneal cavities or cultures of the continuous line RAW 264 responded to activating stimuli (lymphokine-rich spleen cell culture supernates, used either alone or in conjunction with bacterial lipopolysaccharide). Although they often differed quantitatively, qualitative similarities between many of the responses were found, including: response to the same kinds of activating stimuli; induction of cytolytic activity as a consequence of the response; similar kinetics of induction and expression of cytolytic activity; dependence for expression of cytolytic activity on monolayer population density; and a similar spectrum of tumor cell killing, as assayed by 51Cr release during a 16 hr incubation period. In contrast, the ultimate expression of stimulation by these 2 kinds of macrophages was, almost certainly, mechanistically different. This conclusion was reached because tosyl lysine chloromethyl ketone inhibited killing mediated by activated peritoneal macrophages but did not inhibit that which was effected by RAW 264. Thus, it would appear that in-depth analysis will be needed before it can be concluded that responses made to stimulation by normal and continuous cultured macrophages are truly the same or, simply, qualitatively similar. Determination of whether or not continuously cultured macrophages can act as models of the normal process of activation awaits such close scrutiny.