We have developed a sensitive and simple staining method for use in electrophoretic analysis of serum alpha-amylase (EC 3.2.1.1) isoenzymes. The principle of this method is as follows. alpha-Amylase hydrolyzes 4-nitrophenylmaltoheptaoside to generate oligosaccharide, which is then converted to gluconolactone in the presence of oligosaccharide dehydrogenase (no EC no. assigned), with concomitant reduction of 1-methoxy-5-methylphenazinium methylsulfate (1-m-PMS) to 1-m-PMSH. The hydrogen in 1-m-PMSH is then transferred to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to yield formazan. Each of the isoenzymes can then be measured densitometrically. The mean (and SD) values for total amylase, P1, S1, and S2 as determined by this method with sera from 25 healthy adults in fasting were 251 (64), 104 (35), 126 (40), and 22 (11) U/L, respectively. Between-assay CVs (n = 10) for determinations of P1, S1, and S2 were 3.54%, 4.03%, and 7.01%, respectively.