An isocratic reversed-phase high-performance liquid chromatographic method with ultraviolet detection at 227 nm has been validated for the determination of cyclosporin A in human and mouse plasma. The cyclosporin D analog PSC 833 was used as internal standard. Plasma samples were pretreated by liquid-liquid extraction with diethyl ether. A good chromatographic separation between cyclosporin A, the internal standard and two potentially interfering endogenous peaks was achieved using a stainless steel column packed with 5 microm Nova-Pak phenyl material operated at 72 degrees C, and a mobile phase consisting of acetonitrile-methanol-water (20:52:28, v/v/v). The calibration curve for cyclosporin A in human plasma was linear over the tested concentration range of 0.11 to 5.34 microM. Murine plasma samples (200 microl) were diluted up to a total volume of 500 microl with blank human plasma and the concentrations were read from the calibration curve prepared in human plasma. The lower limit of quantitation was 0.11 microM using 500 microl of human plasma and 0.28 microM using 200 microl of mouse plasma. The validation data showed that the assay is sensitive, selective and reproducible for determination of cyclosporin A. The applicability was demonstrated in a pharmacokinetic experiment where mice received oral cyclosporin A.