A simple and precise high-performance liquid chromatographic (HPLC) procedure was developed for the determination of cefsulodin, a new antipseudomonal cephalosporin antibiotic, in plasma. The analytical procedure involved ultrafiltration of samples that were buffered to prevent cefsulodin degradation, followed by injection into an HPLC system, utilizing a C18 reversed-phase analytical column, a mobile phase of acetonitrile-modified aqueous acetate buffer, and a UV spectrophotometric detector. Because of the simplicity of the procedure, the intraassay (or approximately 2%) and interassay (or approximately 3-4%) coefficients of variation were extremely low. Recoveries of drug were essentially quantitative in freshly buffered specimens and in those stored buffered and frozen for nearly 3 months. Calibration curves were rectilinear from the limit of quantification (or approximately 0.2 microgram/ml) to 200 micrograms/ml, as demonstrated by regression correlation coefficients averaging greater than 0.999 during routine analyses.