Class I tRNA synthetases typically have two major domains consisting of a class-defining N-terminal nucleotide binding fold, which contains the active site, and an idiosyncratic C-terminal domain, which in many instances provides for interactions with the tRNA anticodon. Whether the C-terminal domain can function in specific RNA binding when disconnected from the catalytic core is not known. We fused the anticodon binding domain of Escherichia coli methionyl tRNA synthetase to maltose binding protein. This fusion protein and the released, isolated domain are stable and have native-like structural characteristics, as shown by circular dichroism and thermal denaturation studies. Both the fusion protein and the isolated domain bind specifically to a small RNA hairpin oligonucleotide that recapitulates the anticodon stem-loop of tRNAfMet. Neither protein binds to an RNA hairpin with a point mutation in the anticodon trinucleotide. The binding specificity and affinity of these proteins duplicate those of the interaction between methionyl tRNA synthetase and the anticodon stem-loop oligonucleotide. Thus, the anticodon binding domain is functionally independent of the class-defining catalytic core and can be joined to another protein with little change in RNA binding characteristics.