The activation of cellular function following the direct introduction of Ca²⁺ into the cytosol by the use of a Ca²⁺ transporting ionophore has served to confirm the widely held idea that Ca²⁺ has the status of a second messenger in many cell types^1,2. However, this evidence has been obtained largely from the use of a single ionophore, the antibiotic A23187; experiments with X537A, which is another ionophorous antibiotic capable of transporting Ca²⁺ (ref. 3), have failed to show the expected characteristics. For example, histamine release from rat mast cells mediated by X537A is neither dependent on extracellular Ca²⁺ nor prevented by metabolic inhibitors⁴. Ionomycin is a recently described polyether antibiotic produced by Streptomyces conglobatus ATCC 31005, and is active against Grampositive bacteria⁵. The antibiotic action is presumably due to its ionophorous properties, as it extracts Ca²⁺ from an aqueous phase into an organic phase with a stoichiometry of 1:1 (ref. 6). The ionophore is also capable of transporting ⁴⁵Ca²⁺ across biological membranes (our unpublished results). Here we report the application of ionomydn to rat mast cells. We show that ionomycin stimulates mast cell secretion solely through its ability to form a lipid-soluble calcium complex, and thus to convey Ca²⁺ across the hydrocarbon region of the cell membrane.