Ethacrynic acid is an inhibitor of Ca2+-Mg2+-activated ATPases which also inhibits histamine release. By testing analogs of ethacrynic acid, the molecular structural requirements for ATPase inhibition and for inhibition of histamine release were compared. The results indicated that effective inhibition involves several structural features of the molecule. Analogs void of chlorine atoms were ineffective as inhibitors of histamine release and ATPase activity. Inhibition of both ATPase and histamine release requires a sulfhydryl-reactive olefinic bond, but sulfhydryl reactivity alone is not sufficient, as certain analogs which have a high capacity to react with sulfhydryl moieties were not active. Replacement of carboxy by highly ionized moieties like sulfo, rendered the molecules ineffective as an inhibitor of histamine release. Compounds which did not inhibit ATPase activity did not inhibit histamine release. The data indicate that mast cells and basophils require an intact ATPase system for histamine release, and raises the question of whether both ecto and endo ATPases are essential.