Uniformly 15N‐enriched ribonuclease T1 (RNase T1) was obtained from Escherichia coli by recombinant techniques. Heteronuclear 1H, 15N‐shift correlation spectra were recorded utilizing proton detection. Direct 1H, 15N connectivities were established applying the heteronuclear multiple‐quantum coherence technique. Additional 1H, 1H‐TOCSY or 1H, 1H‐NOESY transfer steps allowed for sequential assignments. Nitrogen atoms without directly bonded protons were detected by means of the heteronuclear multiple‐bond correlation experiment. Signals emerging from 15NH and 15NH2 groups were distinguished by heteronuclear triple‐quantum filtering methods. 119 nitrogen resonances out of the expected 127 were assigned unambiguously; in addition, previously obtained proton assignments were extended. Preliminary 1H, 15N NMR investigations were performed on the RNase‐T1–3′GMP inhibitor complex. Results were interpreted with respect to nucleotide binding.