Extracellular adenosine 5′-triphosphate (ATP) induced a characteristic, dose-dependent release of histamine and prostaglandin D2 (PGD2) from rat peritoneal mast cells. The process was relatively slow, non-cytotoxic, maximal at physiological pH and dependent on external calcium. Strontium and barium ions were able to substitute for calcium, although higher concentrations were required for maximal release. Cells stimulated in the absence of calcium progressively lost the ability to respond to subsequent reintroduction of the cation. The secretion of histamine induced by ATP was largely unaffected by the anti-asthmatic drugs disodium cromoglycate and nedocromil sodium but was inhibited by structurally related flavonoids and by cAMP-active drugs. Importantly, the non-hydrolysable guanosine 5′-triphosphate (GTP) analogue, GTP-γ-S, elicited a dose-dependent release of histamine when introduced into mast cells permeabilized with ATP in the absence of external calcium. ATP thus appears to be a useful cell permeabilizing tool with which to study the biochemical processes involved in mast cell activation.