The hydrolysis of chitin (93% N-acetylation) by hydrochloric acid producedN-acetyl-chito-oligosaccharides (NACOs) with low degrees of polymerization (DP)from 2 to 6. The HPLC retention time of NACOs decreased with increased watercontent in the mobile phase. A gradient elution procedure, using acetonitrile to water(v/v) ratio lowered linearly from 80/20 to 60/40 within 60 min, provided optimalresolution and characterization for the NACOs. The natural logarithm of the retentiontime of NACOs correlated linearly with the DP values. Hydrolysis of chitin followeda series-parallel reaction mechanism. At the same time the NACOs with higher DP'swere generated from chitin, and were degraded by hydrochloric acid to form productswith lower DP's. More NACOs with higher DP's were produced under conditions withhigher concentrations of hydrochloric acid and a shorter reaction period. Highertemperatures facilitated the generation of NACOs with lower DP's.