The effect of cycloheximide on the chloroplastic, cytoplasmic and mitochondrial phenylalanyl-transfer RNA synthetases of Euglena gracilis was studied by growing both logarithmic and stationary phase cultures in the presence of the antibiotic. Enzyme activity was measured relative to untreated control cultures. At very low concentrations of cycloheximide (1 microgram/ml), all three log phase enzymes showed an increase in activity of 40-50%. At slightly higher concentrations (2.5 microgram/ml), the phenylalanyl-tRNA synthetase activities were comparable to those of the control cultures. At a cycloheximide concentration of 5 microgram/ml the enzyme activities from stationary phase cultures showed only very slight decreases (5-20%). The cytoplasmic and mitochondrial enzymes behaved similarly in log phase cultures at this concentration. However, the chloroplastic phenylalanyl-tRNA synthetase from log phase cultures treated with 5 microgram/ml cycloheximide showed a marked decrease in activity (70%). A further increase in antibiotic concentration to 10 microgram/ml resulted in significant losses of activity of all three enzymes, from both growth stages. The implications of the data with regard to identification of the site(s) of chloroplast enzyme synthesis are discussed.