The structural requirements for the function of the 3[superscript]' CCA end of E. coli valine transfer RNA in aminoacylation and protein synthesis have been investigated, by studying the activities of 3[superscript]' end tRNA[superscript] Val variants in these processes. These studies show that 3[superscript]' end tRNA[superscript] Val variants can be obtained which are functional in both aminoacylation and later steps of protein synthesis. Replacement of the 3[superscript]' terminal adenosine with either cytosine or uracil yields tRNAs that retain almost full aminoacylation activity (40-50% that of wild type tRNA[superscript] Val). The tRNA[superscript] Val variant with a 3[superscript]'-terminal guanine remains fully chargeable, but is a poor substrate for valyl-tRNA synthetase (VRS). Valyl-tRNA[superscript] Val with 3[superscript]'-CCG is active in in vitro poly(U,G)-directed (Val, Phe) copolypeptide synthesis, whereas valyl-tRNAs with 3[superscript]'-CCC or-CCU are inactive in this process. tRNA[superscript] Val variants at positions 74 and 75 can also be obtained which are functional in both aminoacylation and the later steps of protein synthesis;The determination of the identity elements of E. coli tRNA[superscript] Val has been investigated using two different approaches: first, to determine the sites on tRNA[superscript] Val that are recognized by VRS, aminoacylation kinetic studies of tRNA[superscript] Val variants, accompanied by [superscript]19F NMR spectrosopy of some tRN[superscript] Val variants, are carried out; second, to identify the already-known and additional recognition elements of tRNA[superscript] Val, and determine the negative determinants in noncognate tRNAs that prevent proper recognition of the tRNAs by VRS, the determination of valylation activities of tRNA[superscript] Ala and tRNA[superscript] Phe variants that contain tRNA[superscript] Val identity elements is carried out;These studies show that in addition to the anticodon bases A35 and C36, the discriminator base A73, and the base G20 in the variable pocket are probably recognized by VRS and are identity elements of tRNA[superscript] Val. In addition, the specific conformation of the acceptor stem around base pair U4-A69 may be important for the recognition of tRNA[superscript] Val by VRS. Furthermore, the upper part of the anticodon stem of tRNA [superscript] Val may also contain VRS recognition sites. Our studies suggest that the acceptor stem of E. coli tRNA [superscript] Ala, and the anticodon stem of E. coli tRNA[superscript] Phe contain negative recognition elements that prevent productive interactions between the tRNAs and VRS.