A study has been made of the effect of acetohexamide [N-(p-acetylphenylsulfonyl)-N'-cyclohexylurea] in blood plasma on the creatinine assay procedure based on the Jaffe reaction. An analytical method has been proposed in order to determine the drug in plasma and to clarify the causative relationship between plasma acetohexamide and creatinine values. The method involves the formation of 2, 4-dinitrophenyl (DNP) derivative of the liberated cyclohexylamine from the drug on hydrolysis and its determination by high-pressure liquid chromatography with an ultraviolet detection. It was found that false positive errors in creatinine assay values due to an oral dose of 500 mg of the drug ranged from about 40 to 100 percent in the given subjects with diabetes. The change with time in the concentration of plasma glucose was also described.