The antibiotic chlortetracycline (aureomycin) is shown to be a potent and specific calcium ionophore. The molecule extracts calcium from an aqueous environment into a bulk organic phase in a pH-dependent manner and with a stoichiometry indicative of a 1:1 complex. The antibiotic has little affinity for other alkaline earth ions or alkali metal ions in this system. The ionophore transports calcium from one aqueous phase to another across an organic solvent barrier and induces a flux of calcium into multilamellar vesicles. The potential use of the compound in examining the role of calcium in the modulation of cellular function is discussed.