Substrates commonly used for the determination of amylase activity include potato starch, corn starch and dye-labeled starch. Determination of the amylase activity of serum using these different starches has shown that the measured value varies depending upon the ratio of isoamylases present, namely between pancreatic amylase (P-type) and salivary amylase (S-type), contained in the serum. With corn starch as substrate, the P-type dominant serum exhibited an apparently higher value than the S-type dominant serum. In the use of blue-starch which is employed as a chromogenic method, the P-type dominant serum gave a higher value than the S-type dominant serum. Red-starch which is also used as a chromogenic method, however, did not cause the P-type dominant serum to show such a high level of amylase activity as blue-starch. These differences in amylase activity can be also shown by determining the Km values of pancreatic amylase and salivary amylase using these substrates. Thus, corn starch and blue-starch showed smaller Km values to pancreatic amylase than to salivary amylase. They were thus proved to have a strong affinity for pancreatic amylase. In contrast, potato starch, red-starch and glycogen had good affinity for salivary amylase. In pancreatic disease in which pancreatic amylase is increased without much elevation in the total amylase level in the serum, it might be possible to detect the abnormality of pancreatic amylase activity if either corn starch or blue-starch is used as a substrate for measurement of the serum amylase activity.