Euglena gracilis chloroplast protein synthesis elongation factor G (EF-Gchl) has been purified to about 80% homogeneity by a two-step procedure which removes all traces of the cytoplasmic and mitochondrial translocases (EF-2 and EF-Gmt). The purification scheme generally results in approximately 130-fold purification with 20% recovery of the total EF-G activity present in whole cell extracts. The Euglena EF-Gchl is a monomeric protein with a molecular weight of approximately 85,000. As observed for all translocases to date, the activity of Euglena EF-Gchl is inhibited by treatment with low concentrations of N-ethylmaleimide, suggesting that a free sulfhydryl group is required for catalytic activity. Treatment with 3 microM fusidic acid results in a 50% inhibition of Euglena EF-Gchl activity and of the EF-G activity present in Chlamydomonas reinhardtii. About 10-fold higher concentrations of this antibiotic are required to inhibit the mitochondrial EF-G of Euglena and Escherichia coli EF-G to the same extent. Yeast mitochondrial EF-G is clearly distinguishable from the other organellar translocases tested, requiring 1 mM fusidic acid for 50% inhibition. Fusidic acid also inhibits the cytoplasmic translocases from yeast, wheat germ, and Euglena, although a wide range of sensitivities is observed. When antiserum raised against highly purified Euglena EF-Gchl is used to inhibit enzymatic translocation, a low degree of cross-reaction of the antiserum with Chlamydomonas EF-G and with E. coli EF-G is observed. The EF-G activity present in spinach is very slightly inhibited by the antiserum, whereas that of yeast is not affected. The mitochondrial and cytoplasmic translocases of Euglena are also unaffected by the antiserum against EF-Gchl. The evolutionary implications of these observations are discussed.