In 1984, our laboratory reported the characterization of the first misacylated tRNA missense suppressor, a mutant Escherichia coli lysine tRNA with a C70 to U base change in the amino acid acceptor stem. We suggested then that the suppressor tRNA, though still acylated to a large extent with lysine, is partially misacylated with alanine. The results reported in this article demonstrate that is the case both in vitro and in vivo. For the in vitro studies, the mutant tRNA species was isolated from the appropriate RPC-5 column fractions and shown to be acylatable with both lysine and alanine. For the in vivo demonstration, use was made of a temperature-sensitive alaS mutation, which results in decreasing acylation with Ala as the temperature is increased, resulting ultimately in lethality at 42 degrees C. The alaSts mutation was also used to demonstrate that the ability of the same missense suppressor, lysT(U70), to suppress a trpA frameshift mutation is not affected by the Ala-acylation deficiency. We conclude that the misacylation and altered decoding are two independent effects of the C70 to U mutation in tRNA(Lys). The influence of an alteration in the acceptor stem, which is in contact with the large (50S) ribosomal subunit, on decoding, which involves contact between the anticodon region of tRNA and the small (30S) ribosomal subunit, may occur intramolecularly, through the tRNA molecule. Alternatively, the U70 effect may be accomplished intermolecularly; for example, it may alter the interaction of tRNA with ribosomal RNA in the 50S subunit, which may then influence further interactions between the two subunits and between the 30S subunit and the anticodon region of the tRNA. Preliminary evidence suggesting some form of the latter explanation is presented. The influence of a single nucleotide on both tRNA identity and decoding may be related to the coevolution of tRNAs, aminoacyl-tRNA synthetases, and ribosomes.