In this procedure, reversed-phase liquid chromatography is used to measure cyclosporine, a fungal metabolite with immunosuppressive activity, in human serum. With a gradient elution, the retention times for cyclosporine and cyclosporin D were 14.1 and 15.7 min, respectively. Ultraviolet absorbance at 215 nm was used to detect cyclosporine; this wavelength improved assay accuracy without decreasing sensitivity, as compared with detection at 205 nm, which is near the absorption maximum. The major advantage of our procedure is the clean-up method, which involves use of disposable extraction columns. This extraction is simple and requires only 10 to 15 min per sample. Results by radioimmunoassay for cyclosporine were unpredictably greater than those measured by the present method. Dosing guidelines for cyclosporine need re-evaluation, based on more specific assay methods.