A single G3:U70 base pair in the acceptor helix is the major determinant of alanine acceptance in alanine transfer RNAs. Transfer of this base pair into other transfer RNAs confers alanine acceptance. A G3:C70 substitution eliminates alanine acceptance in vivo and in vitro. In this work, a population of mutagenized G3:C70 alanine tRNA amber suppressors was subjected to a selection for mutations that compensate for the inactivating G3:C70 substitution. No compensatory mutations located in the acceptor helix were obtained. Instead, a U27:U43 substitution that replaced the wild-type C27:G43 in the anticodon stem created a U27:U43/G3:C70 mutant alanine tRNA that inserts alanine at amber codons in vivo. The U27:U43 substitution is at a location where previous footprinting work established an RNA-protein contact. Thus, this mutation may act by functionally coupling a distal part of the tRNA structure to the active site.