S. hygroscopicus has been shown to produce a second antibiotic substance, hygromycin B. Isolation of hygromycin B was accomplished by chromatography on a cation-exchange resin, carbon adsorption and countercurrent distribution. Properties and partial characterization of the new antibiotic have been described. Consideration of the consequences of multifunctionality of the catalytically active site of a-chymotrypsin and of a class of its representative specific substrates led to the conclusion that under comparable conditions the rate of the a-chymotrypsincatalyzed hydrolysis of a-N-(nicotinyl-1-oxide)-L-phenylalanine methyl ester should be substantially greater than that of a-N-benzoyl-P-(4-pyridyl-l-oxide)-L-alanine methyl ester. This prediction has been verified by the observation that the relative rate of hydrolysis of the former specific substrate is ca. 200 times greater than that of the latter.