A simple method for the assay of histatins in human saliva by high-performance liquid chromatography has been developed. The histatins in saliva were extracted with 0.1 M HCl-methanol, and histatins 1, 3, 5, and 6 were separated and concentrations were determined by reversed-phase chromatography. This simple method enabled the determination of levels of individual histatins from the saliva of normal subjects. The average concentration of histatins 1, 3, 5, and 6 in parotid saliva collected from 26 healthy volunteers aged from 20 to 30 years were 11.25 +/- 5.65, 8.15 +/- 3.08, 7.67 +/- 3.12, and 1.56 +/- 0.53 mumol/l (mean +/- S.D.), respectively.