Bombyx mori glycyl-tRNA synthetase (GRS) was expressed as the full length protein and as N-terminally and C-terminally truncated forms. The intact enzyme and forms with deletions of 12, 27, 46, and 55 N-terminal residues were expressed, purified, and characterized. All were active, having 15-25% of both pyrophosphate exchange activity and aminoacyl-tRNA synthetase activity compared to wild type enzyme. Active site titration indicated that this difference in activity was not the result of production of inactive enzyme. Sedimentation and gel filtration experiments indicate that the N-terminally deleted forms and the wild type enzyme were dimers. Deletion of 55 N-terminal residues did not result in significant effects on the Michaelis constants for ATP, glycine, or tRNA, while deletion of 108 N-terminal residues and two internal 64- and 200-residue deletions generated inactive forms. Five forms with C-terminal deletions of 24, 37, 59, 162, and 327 amino acid residues were soluble and intact but lacked detectable pyrophosphate exchange activity or aminoacyl-tRNA synthetase activity. The C-terminal sequence may be required for catalysis or to maintain a stable structure. Zone electrophoresis demonstrated the wild type enzyme bound both tRNA(Gly) and noncognate tRNA(Ala). Deletion of 55 N-terminal residues resulted in altered binding of tRNA(Gly) and eliminated binding of tRNA(Ala). The first 55 N-terminal residues are not essential for catalysis, dimerization, or substrate binding in aminoacylation but are required for RNA binding not associated with aminoacylation.