Family 18 chitinases such as chitinase B (ChiB) from Serratia marcescens catalyze glycoside hydrolysis via a mechanism involving the N-acetyl group of the sugar bound to the -1 subsite. We have studied the degradation of the soluble heteropolymer chitosan, to obtain further insight into catalysis in ChiB and to experimentally assess the proposed processive action of this enzyme. Degradation of chitosans with varying degrees of acetylation was monitored by following the size-distribution of oligomers, and oligomers were isolated and partly sequenced using (1)H-NMR spectroscopy. Degradation of a chitosan with 65% acetylated units showed that ChiB is an exo-enzyme which degrades the polymer chains from their nonreducing ends. The degradation showed biphasic kinetics: the faster phase is dominated by cleavage on the reducing side of two acetylated units (occupying subsites -2 and -1), while the slower kinetic phase reflects cleavage on the reducing side of a deacetylated and an acetylated unit (bound to subsites -2 and -1, respectively). The enzyme did not show preferences with respect to acetylation of the sugar bound in the +1 subsite. Thus, the preference for an acetylated unit is absolute in the -1 subsite, whereas substrate specificity is less stringent in the -2 and +1 subsites. Consequently, even chitosans with low degrees of acetylation could be degraded by ChiB, permitting the production of mixtures of oligosaccharides with different size distributions and chemical composition. Initially, the degradation of the 65% acetylated chitosan almost exclusively yielded oligomers with even-numbered chain lengths. This provides experimental evidence for a processive mode of action, moving the sugar chain two residues at a time. The results show that nonproductive binding events are not necessarily followed by substrate release but rather by consecutive relocations of the sugar chain.