We have demonstrated efficient protein synthesis in a cell-free system from the bloodstream form of Trypanosoma brucei. This system was able to translate endogenous mRNA, added mRNA, or (apparently at much lower efficiency) three synthetic RNA transcripts lacking 5' mini-exon and 3' poly(A) sequences. Translation was resistant to chloramphenicol and greater than 95% inhibited by low concentrations of anisomycin and puromycin, but only partially inhibited by cycloheximide. Variant surface glycoprotein synthesized from endogenous mRNA was sensitive to endoglycosidase H, indicating the co-translational glycosylation potential of the system. Two proteins translated ab initio from the corresponding in vitro-transcribed RNAs showed no evidence of signal sequence cleavage or glycosylation. Efficient processing occurred when the same RNAs were translated in a rabbit reticulocyte lysate supplemented with canine pancreatic microsomes but not with trypanosome microsomes. (C) 1991 Elsevier Science B.V. All rights reserved.