The effect of various tRNAs on protein synthesis was investigated using a tRNA-dependent cell-free system from Ehrlich ascites cells. Ascites cell tRNA and rabbit liver tRNA promoted efficient translation of globin mRNA, oviduct mRNA and encephalomyocarditis (EMC) viral RNA. Reticulocyte tRNA participated efficiently only in the translation of globin mRNA; the translation of oviduct mRNA and EMC viral RNA in the presence of reticulocyte tRNA resulted in the synthesis of relatively few large mature proteins and the accumulation of discrete, smaller polypeptides. Isoaccepting tRNA species may be required for the synthesis of ovalbumin EMC viral protein (but not Hg) are probably functionally absent in reticulocyte tRNA, causing a premature, nonrandom termination of synthesis of these proteins. This provides preliminary evidence that variations in tRNA populations, frequently observed between different cell types, are large enough to define and perhaps regulate the proteins that the cell is capable of synthesizing.