Hydrogen peroxide was generated from creatinine by the sequential enzyme reactions of creatinine amidohydrolase, creatine amidinohydrolase and sarcosine oxidase. Hydrogen peroxide was, in turn, used stoichiometrically for the condensation of 4-aminoantipyrine and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine catalyzed by horse-radish peroxidase, resulting in the formation of quinone dye with maximum absorbance at 546 nm. The optimized assay conditions for the enzymatic determination of creatinine in a HITACHI 7250 autoanalyzer was established. This system, which requires less than 5 μl of sample, was found to be the most economical for laboratories equipped with autoanalyzers.