A method for separation of serum (pseudo) cholinesterase isozymes was studied in order to take advantage of it for clinical research. By employing modifications on a previously reported method, analytical time was shortened to a half (1.5 days) of the original method without abolishing qualitative and quantitative accuracy. Thus, the present method facilitated its clinical application for the study of this isozyme. A normal pattern of the isozyme in Japanese in this method was determined by the analysis of sixteen normal subjects, which appeared to be very consistent in each individual under the present conditions. The distribution of relative activities of respective isozymes measured on a densitogram coincided well with that measured calorimetrically. Abnormalities of the zymogram were newly found in patients with liver cirrhosis and metastatic liver cancer, which seemed to be characteristic to the respective diseases. Isozyme patterns in the liver and ascites were also measured and compared with those in serum.