The cyst wall of Entamoeba invadens (Ei), a model for the human pathogen Entamoeba histolytica, contains chitin, which is a homopolymer of beta-1, 4-linked N-acetyl-glucosamine (GlcNAc). In fungi and in bacteria that make nodulation factors, chitin deacetylases make chitosan, which is a mixture of GlcNAc and glucosamine and so has a positive charge. The activity of an Ei chitin deacetylase was revealed by a 3-4-fold increase in released GlcNAc when deproteinated cyst walls were chemically acetylated prior to treatment with a commerical chitinase. Because this chitinase releases GlcNAc but not GlcN, increases in released GlcNAc after acetylation suggested the presence of chitosan in Ei cyst walls. Five putative Ei and Eh chitin deacetylase genes resembled those of fungi and bacteria. A predicted Eh chitin deacetylase matched closely the three-dimensional structure of a Bacillus subtilis peptiodglycan deacetylase. A recombinant Eh chitin deacetylase, expressed in Saccharomyces cerevisiae, deacetylated chitooligosaccharides in vitro. These results are consistent with the idea that Ei chitin deacetylases modify chitin to produce chitosan in the Ei cyst wall.