In this study, we identify a subset of nucleotides that specify aminoacylation of tRNA(Pro) by Escherichia coli proline tRNA synthetase in vitro. Twenty-two tRNA(Pro) variants were prepared by in vitro transcription and their efficiency of aminoacylation with proline (kcat/KM) was measured. From this analysis, we conclude that recognition elements for tRNA(Pro) aminoacylation by ProRS are located in at least three domains of the tRNA molecule. The largest decreases in the kinetic parameters for aminoacylation resulted from single substitutions at position G72 of the acceptor stem and position G36 of the anticodon. Anticodon nucleotide G35 and position A73 in the acceptor stem were also identified as major recognition elements. Moreover, bases that are believed to be important for maintaining the tertiary structure of the tRNA (G15 and C48) appear to be important for efficient recognition of tRNA(Pro) by ProRS in vitro.