Determinants for the identities of tRNAs are located in the acceptor stem and, commonly, in the anticodon as well. Although the anticodon is an important determinant for the identity of methionine tRNA, RNA microhelices whose sequences are based on the acceptor stem alone can be aminoacylated by the class I methionyl-tRNA synthetase. We show here that specific nucleotide substitutions in a microhelix significantly reduced its rate of aminoacylation. In contrast, affinity coelectrophoresis analysis showed that microhelix binding to the enzyme was not significantly affected by the same substitutions. These and additional experiments and considerations imply that specific determinants for microhelix aminoacylation are needed for orientation of the acceptor stem in the transition state of catalysis rather than for enhanced binding interactions. The effect of linking together acceptor stem interactions with those in the anticodon, as occurs in the whole tRNA molecule, was also evaluated. This analysis showed that linkage results in some of the favorable acceptor stem and anticodon interactions being used to offset the free energy cost of straining the structure of the enzyme-tRNA complex.