1. Transfer-RNA of high purity is prepared from intact Euglena gracilis cells. Chromatography on benzoylated DEAE-cellulose columns succeeds in the separation of chloroplast tRNA Leu from cytoplasmic tRNA Leu . 2. The chromatographic separation of tRNA from green mixotroph or autotroph cells yield one major and one minor peak of cytoplasmic tRNA Leu and at least two tRNA Leu peaks with prokaryotic acceptor specificity. The tRNA preparation of a plastid mutant of E . grabilis does not contain tRNA Leu species of the prokaryotic type, in contrast to the tRNA prepared from dark grown cells. 3. The prokaryotic (chloroplast) tRNA Leu fractions effectively accept 14 C-Leucine by means of enzyme preparations from isolated chloroplasts and isolated mitochondria of E. gracilis and the total enzyme fraction of A. nidulans . Cytoplasmic leucyl-tRNA synthetase of E. gracilis is unable to charge plastid tRNA Leu . 4. The tRNA prepared from A. nidulans can be separated into 5 tRNA Leu fractions. They are charged by separated chloroplast leucyl-tRNA synthetase at the same extent as by the homologous A. nidulans enzyme. 5. Our results demonstrate a strict aminoaeylation specificity, in heterologous combinations, between the tRNA Leu and leucyl-tRNA synthetases of A. nidulans or of the prokaryotic organelles versus the cytoplasmic components of E. gracilis . Our results further show that chloroplast tRNA Leu or its cognate enzyme can be replaced by the components of A. nidulans; cytoplasmic tRNA is exchangeable against total tRNA prepared from intact cells of a plastid mutant of E. gracilis . 6. Chloroplast tRNA Leu and chloroplast leucyl-tRNA synthetase increase during the light-dependent greening process of dark-grown E. gracilis . The induced enzyme synthesis clearly precedes chlorophyll formation and chloroplast tRNA Leu accumulation of approximately 6 h under our experimental conditions. It is suggested that the lag period reflects a period necessary for the formation of thylakoid membranes as prerequisite for the plastid RNA polymerase to become an active enzyme.