Chromatographic and functional properties of tyrosine isoaccepting transfer ribonucleic acids (tRNAs) from placenta and HeLa cells were analyzed and compared. RPC-5 chromatography separated four major isoacceptors from each source, with those from HeLa cells eluting generally later than those from placenta. There was some overlap: HeLa tRNA1Tyr eluted in a position between placenta tRNA3Tyr and tRNA4Tyr; and HeLa tRNA2Tyr and placenta tRNA4Tyr eluted in similar positions with the HeLa isoacceptor eluting slightly later than the placental isoacceptor. Thus there are no isoacceptors common to both sources. The function of the individual isoacceptors was compared in a rabbit-reticulocyte, cell-free, protein-synthesizing system for both the rate of incorporation of tyrosine into the polypeptide chain and the site of incorporation in alpha-globin. Two isoacceptors were compared simultaneously in the same reaction, and overlapping comparisons were made to relate each isoacceptor to all the others. There were no significant differences in the rates of incorporation among the isoacceptors, nor were there any differences in the sites of incorporation. All eight isoacceptors donated tyrosine equally well into the three tyrosine containing tryptic peptides of alpha-globin. Whatever the structural differences among the tyrosine isoacceptors are, they do not affect the function of the tRNA in this protein-synthesizing system.