Ribonuclease T 1 is highly specific for the guanylic acid residue in polyribonucleotides. To clarify the origin of the substrate specificity, the interaction sites of guanylic acid with ribonuclease T 1 were investigated by the use of 15 N-NMR. 95% 15 N-enriched guanosine-3′-phosphate was prepared and mixed with purified ribonuclease T 1 . 15 N-NMR spectra of the mixtures at different concentrations were obtained and compared with that of the 15 N-enriched substrate alone. Upon complex formation, a 15 N signal assigned to the amino group nitrogen at position 2 of guanine shifted and was significantly broadened, suggesting a strong interaction with the enzyme through the amino group. This observation is consistent with the results of studies on the substrate specificity by chemical modification. Nuclear Overhauser effects of signals assigned to N-7 and N-3 were also changed, but no shift was observed. The observations do not support the occurrence of protonation at N-7 upon complex formation, which was previously proposed.