Studies have been conducted on the mechanisms of release of histamine from isolated mast cells by a purified basic protein of neutrophilic leukocytes. Release was inhibited in the cold, by 2,4-dinitrophenol (DNP) (5 × 10-5) in the absence of glucose, by iodoacetic acid (2 × 10-3), and by heating the cells to 45°C. Organophosphorus esterase inhibitors also prevented release of histamine when added simultaneously with the neutrophilic basic protein. Mast cells activated in the presence of DNP inhibitor and then washed released histamine upon addition of glucose. This release was not affected by diisopropylfluorophosphate, but was inhibited by chelation. Mast cells exposed at 37°C to basic protein in the presence of DNP for varying times, and then washed and added to glucose-containing medium, demonstrated increasing inability to release histamine over a 30-min period of initial exposure. These cells would not respond to fresh addition of the basic protein. In contrast, cells held at 10°C in the presence of basic protein would still respond after washing to the fresh addition of basic protein. The data provide evidence for distinct intracellular steps in the process of activation of mast cells and release of histamine.