The mechanism of Entamoeba histolytica cyst cell wall synthesis is not well understood. Previous research has shown that cyst-like structures formed in the presence of chitin synthase cofactors (Mg2+, Mn2+, and Co2+) resist 1% sodium dodecyl sulfate lysis (RCLS), whereas those formed in the absence of cofactors (CLS) do not, and trophozoites are immediately destroyed. This suggests that E. histolytica is able to synthesize chitin, initiating a differentiation process under axenic conditions. To test this hypothesis, polysaccharide hydrolysates from E. histolytica trophozoites, CLS, or RCLS were analyzed with high-performance liquid chromatography. The major components found in all 3 preparations were N-acetylglucosamine (NAG) and glucose (GLC), with RCLS possessing 129 and 180 times more NAG and 2.4 and 2.0 more GLC than trophozoites and CLS, respectively. After 36 hr of incubation with chitinase (16 U/ml) in a hypotonic medium (50 mOsm/kg), 68% of RCLS was lysed, and 100% lost affinity for calcofluor white M2R. The RCLS polysaccharides bound wheat germ agglutinin and appeared as long and thin or short and thick fibers. Accordingly, Mg2+, Mn2+, and Co2+ stimulated E. histolytica to synthesize a chitin-like material.