The trinucleotide CCA sequence is present at the 3' terminus of all mature tRNAs. Despite this high degree of conservation, we have been able to prepare in vitro transcripts of Escherichia coli tRNA(Val) with altered 3' termini that are readily aminoacylated and can function in polypeptide synthesis. Replacement of the 3'-terminal adenosine with either cytidine or uridine yields a tRNA(Val) variant that retains almost full aminoacylation activity, having specificity constants (Vmax/Km) 40-50% that of wild-type tRNA(Val). The tRNA(Val) variant with a 3'-terminal guanosine remains fully chargeable but is a poor substrate for valyl-tRNA synthetase, largely as the result of a decrease in the catalytic constant. End-group analysis revealed the absence of adenosine at the 3' end of the tRNA(Val) mutants and identified the nucleotide expected from the sequence of the DNA template as the predominant 3'-terminal residue; Val-cytidine was isolated from the aminoacylated C76 mutant. Val-tRNA(Val) with 3'-CCG is active in poly(U,G)-directed (Val, Phe) copolypeptide synthesis, whereas the tRNA(Val) mutants terminating in 3'-CCC and 3'-CCU, which are readily aminoacylated, are inactive. The differential effects of nucleotide substitution on aminoacylation and polypeptide synthesis suggest that the universally conserved 3'-CCA end of tRNAs is monitored at two or more steps in protein synthesis that have different nucleotide recognition specificities.