Micrococcus luteun (M. lysodeikticus) labeled with Remazol brilliant blue R (blue ML) was prepared as a novel substrate for the colorimetric assay of lysozyme. The treatment of the labeled substrate with lysozyme resulted in the release of soluble blue products which can be easily measured spectrophotometrically at 600nm. The blue color was most efficiently released at pH 7 and ionic strength of 0.2 on incubation with hen lysozyme at 40°C. A new colorimetric method for the assay of lysozyme using this substrate was developed. The assay system gave a linear dose-response curve, and as little as 0.1μg of human lysozyme (1μg/ml, 100μl) can be detected. The present method is more convenient and reproducible than the conventional lysozyme assay with bacterial cells. Application of the system to the determination of lysozyme in human serum is described.