We assayed creatine kinase (EC 2.7.3.2) activity in normal serum and in specimens obtained from patients with high serum creatinine by four automated methods: (a) the Technicon SMA 12/60, (b) the Technicon AutoAnalyzer Ii, (c) the Abbott Bichromatic Analyzer, and ( the Aminco Rotochem. Activity blanks were assayed with the AutoAnalyzer II system. With the colorimetric assay, which measures the amount of creatine produced, we obtained high blank values (up to 254 U/liter) for patients with increased serum creatinine, while those with normal serum creatinine showed slight to moderate blank values (up to 35 U/liter). Patients with abnormally high values for serum creatinine should have their creatine kinase activity assayed with use of either a blank correction or by a method that does not measure the amount of creatine produced. Paper chromatography and thin-layer chromatography of selected sera indicate that endogenous creatine is the major source of interference in the colorimtric procedure.