Synthetic peptides were used to probe O- and N-glycosylation reactions in cell-free systems of the parasitic protozoa Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma brucei brucei. O-Glycosylation of the peptide Pro-Tyr-Thr-Val-Val was observed with lysates from all organisms. However, the spectrum of sugars transferred from their respective nucleotide or dolichol-phosphate derivatives to the peptide varied greatly according to the parasite. N-glycosylation of the peptides N-Bz-Asn-Gly-ThrNH2 and DNP-Arg-Asn-Ala-Thr-Ala-ValNH2 by exogenous radioactive dolichol-pyrophosphate linked oligosaccharide donors was observed only when lysates of T. gondii or T. b. brucei were used, but not in P. falciparum. To assay for endogenous N-glycosylation donors, the radiolabeled tripeptide [3H]Ac-Asn-Gly-ThrNHMe was used as acceptor. The peptide was N-glycosylated only by T. gondii and T. b. brucei preparations. Only in these latter two parasites dolichol-cycle mannosyltransferase activity was demonstrated by the elongation of exogenous radiolabeled dolichol-PP-chitobiose. The data substantiate the occurrence of protein O-glycosylation in parasitic protozoa and the exceptional absence of protein N-glycosylation in the asexual intraerythrocytic stage of the malaria parasite, P. falciparum.