We describe an HPLC method for quantifying creatinine, separating the analyte from other compounds in serum and urine by cation-exchange chromatography and measuring its absorbance at 234 nm. The precision of the method (CV) varied from 2.9% (mean creatinine concentration, 31 mumol/L) to 1.7% (361 mumol/L) within a series of assays and from 3.9% (34 mumol/L) to 2.4% (391 mumol/L) between series. A comparison with the Jaffé method, as performed with a Technicon SMA analyzer, gave the regression line yHPLC = 1.00xJaffé - 12.0 (n = 141, r = 0.998, and Syx = 19). Results also are comparable with those of an enzymatic method, if the enzymatic method is standardized with a serum-based standard when serum samples are measured. An aqueous standard has to be used for enzymatic determination of creatinine in urine.