Glycosomes are intracellular, membrane-bound microbody organelles of trypanosomes and leishmania. Nine glycolytic enzymes are the major protein components of the glycosomes of Trypanosoma brucei long-slender bloodstream forms. Glycosomal proteins are believed to be synthesized in the cytoplasm and inserted across the glycosomal membrane posttranslationally. We have developed an in vitro protein import assay for the study of glycosomal biogenesis in T. brucei. All nine glycosomal glycolytic enzymes were detectable by immunoprecipitation and gel analysis of radiolabeled products derived from in vitro translation of total mRNA. Radiolabeled translational products were incubated with purified glycosomes isolated from bloodstream forms and digested with protease to remove proteins not imported into glycosomes. Gel analysis of reisolated glycosomes revealed that glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and 3-phosphoglycerate kinase (PGK) (EC 2.7.2.3) were apparently imported intact into the glycosome. Specificity of the protein import assay was verified by using translational products derived from cloned genes encoding T. brucei glycosomal PGK and its 95% homologous cytosolic isozyme. Glycosomal PGK was inserted into the glycosome in vitro with a 27.6% efficiency, but no imported cytosolic PGK was detectable. Preliminary data suggest that certain sequences between the N terminus and residue 123 may be important for import of glycosomal PGK. Our assay, combined with the potential use of genetically altered substrate proteins, may provide the opportunity to explore the recognition systems involved in glycosome biogenesis.