Polymyxin B produced a dose-dependent release of histamine from rat peritoneal mast cells. The response was non-cytotoxic and extremely rapid. The release was maximal in the presence of extracellular calcium ions but a substantial component persisted in the absence of the cation. This component was virtually abolished by pretreatment with the ionophore A23187 in the presence of a chelating agent and thus probably reflected the mobilization of intracellular stores of calcium. Hamster peritoneal cells also responded to polymyxin but were less reactive than those of the rat. Mouse peritoneal cells and guinea-pig lung mast cells responded only at very high concentrations. These results further emphasize the functional heterogeneity of mast cells from different sources. Histamine release from rat peritoneal mast cells was inhibited in dose-dependent fashion by cAMP analogues and anti-allergic drugs. The inhibitory effect varied inversely with the magnitude of the control secretion. With the exception of disodium cromoglycate, the test drugs were essentially equiactive in the presence and absence of extracellular calcium. These results are discussed in terms of the possible mode of action of these compounds.