A high-performance liquid chromatographic method was developed for the determination of carnitine and 11 acylcarnitines (acetyl-, propionyl-, butyryl-, isovaleryl-, hexanoyl-, octanoyl-, decanoyl-, lauroyl-, myristoyl-, palmitoyl-, and stearoylcarnitine) in human urine. Urine samples were treated with a disposable cation-exchange column to extract carnitine and acyl-carnitines using cyclopentanoylcarnitine as an internal standard. The extracted carnitine and acylcarnitines were reacted with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone as a fluorescence derivatization reagent and were separated and detected by high-performance liquid chromatography with gradient elution. Calibration curves gave good linearities at the individual assay range. Analytical recoveries were 70-76% for palmitoylcarnitine and stearoylcarnitine, and were more than 96% for carnitine and the 9 other acylcarnitines. The lower limits of quantitation were 103 nmol/ml for carnitine, 19 nmol/ml for acetylcarnitine, and 0.5-10.5 nmol/ml for other acetylcarnitines. Precision and accuracy including within- and between-run were < or = 19% and -17 approximately +13% at the lowest concentrations of the individual assay ranges. The coefficient of correlation between this method and the radioenzymatic method was found to be 0.988 for the measured concentration of carnitine. We concluded that the present method can be used to measure carnitine and acylcarnitines in human urine.