A sensitive method for the specific determination of creatinine in whole blood, plasma, and urine with high precision and accuracy is described. Samples were deproteinized by addition of acetonitrile and analyzed by high-performance liquid chromatography using a cation-exchange column with a mobile phase of 9% acetonitrile in 40 mM ammonium phosphate (pH 4.0). The recoveries of creatinine added to blood and plasma were almost complete, ranging from 99 to 101%. The coefficients of variation were very small, 1.6% for blood and plasma and 1.5% for urine. Samples can be assayed in 11-min intervals subsequent to the initial injection. As little as 2 microliter of blood or plasma or 0.02 microliter of urine is sufficient for chromatographic analysis. The present method was successfully used for the accurate measurement of small arterial-venous differences of creatinine concentrations in blood across body organs and showed that in the sheep creatinine is produced in the muscles and is excreted by the kidneys. The method is also suitable for routine analysis of creatinine in clinical laboratories.