Coumermycin A1 is an antibiotic isolated from Streptomyces hazeliensis var. hazeliensis nov. sp. as a sodium salt which exhibits antistaphylococcal activity. A sensitive and selective high-performance liquid chromatographic method was developed for the determination of the compound and three known homologues which are extracted from plasma buffered to pH 6.5 into methyl-tert.-butyl ether-2-propanol (97.5:2.5), the residue of which is dissolved in the mobile phase and analyzed by automated reversed-phase high-performance liquid chromatography using UV detection at 330 nm for quantitation. Novobiocin is used as the internal standard. The method was used to determine the plasma concentration--time profile of coumermycin A1 in the dog following a single intravenous administration of a 12 mg/kg dose of a solubilized dosage form of the bulk drug substance.