A new determination method of pemoline in plasma, plasma water, mixed saliva, and urine using high-performance liquid chromatography was developed. The detection limit of pemoline using 0.2 ml of the sample was 0.02 microgram/ml in plasma, plasma water, saliva, and urine. The recoveries of pemoline added to plasma, saliva, and urine (each 6 micrograms/ml) were more than 98%. The coefficients of variation of within-run and between-run precisions for 5 concentrations of pemoline in plasma were less than 5 and 6%, respectively. The elimination half-lives of pemoline obtained from the plasma concentration-time curves after a single oral administration of pemoline in two subjects were 6.7 and 10.3 h. Mean values of the ratio between saliva and plasma total concentration in two subjects were 0.55 and 0.64, and mean values of plasma protein binding percent were 35.7 and 25.2%, respectively. The saliva concentrations were in proportion to the plasma unbound pemoline concentrations at simultaneous samplings, and the ratios between saliva and plasma unbound concentration were about 0.9 in two subjects. Usefulness of saliva in the estimation of plasma protein-unbound pemoline concentration was noted.