The mechanism of macrophage activation was studied using three activating substances, guinea pig macrophage activation factor (MAF), lipopolysaccharide (LPS) and muramyl dipeptide (MDP). Guinea pig peritoneal exudate macrophages were activated to exhibit the accelerated glucose consumption in response to these activating substances. Calmodulin-specific inhibitors, trifluoperazine and No. 233, inhibited macrophage activation with MAF and LPS, while these inhibitors did not affect the activation with MDP. Ca2+ uptake into macrophages was enhanced in MAF-treated macrophages, but LPS and MDP did not affect the Ca2+ uptake. Methylamine and ethylamine, inhibitors of transglutaminase-dependent protein internalization into cells and/or of lysosomal enzymes, effectively inhibited the activating effect of LPS, but not those of MAF and MDP. These results suggest that Ca2+ and calmodulin play a role in macrophage activation with MAF, and neither transglutaminase-dependent internalization nor lysosomal enzymes participate in the activation process. In case of LPS, internalization into cells would be necessary for its activating effect. The processing of the contrary, since the activating effect of MDP was not affected by any of these inhibitors, the mechanism of activation with MDP remains obscure. Thus, the mechanisms of macrophage activation with MAF, LPS and MDP appear to be different from each other.