Sequential incubation of a mixed membrane fraction isolated from Entamoeba histolytica trophozoites with the nonionic detergents Brij 35 and Igepal CA-630 rendered a soluble fraction with the ability to transfer N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to dolichol phosphate to form a lipid saccharide that was identified as a mixture of dolichol-P-P-GlcNAc and dolichol-P-P-(GlcNAc)2 as follows. (a) The reaction occurred only in the presence of exogenously added dolichol phosphate and was strongly inhibited by tunicamycin and amphomycin; (b) Over 90% of the aminosugar moiety of the lipid saccharide was released by mild acid hydrolysis and was identified as a mixture of GlcNAc and diacetylchitobiose [(GlcNAc)2]; (c) Time course experiments revealed that dolichol-P-P-(GlcNAc)2 accumulated at the expense of a parallel decrease in dolichol-P-P-GlcNAc revealing the tandem operation of UDPGlcNAc:dolichol-P GlcNAc-1-P transferase and UDPGlcNAc:dolichol-P GlcNAc transferase. Mg2+ and to a lower extent Mn2+ were required for catalytic activity and were optimal at 2.5 mM and 1.25 mM, respectively. Common phospholipids with different head groups failed to increase catalytic activity and phosphatidylglycerol was inhibitory. At low concentration, nucleotides such as ATP, GMP and GTP brought about stimulations of 24-54% but higher concentrations were inhibitory. Others were inhibitory at all concentrations the strongest being those containing a uridine base.