A regular dependence of the (1)J(NC’) coupling constant on the nature of amide group hydrogen bonding is observed in the model compound, N-acetylglycine. Hydrogen bonding of the amide oxygen increases the coupling constant, whereas hydrogen bonding of the amide hydrogen decreases it. This establishes the (1)J(NC’) coupling constant as a useful probe for amide group hydrogen bonding in proteins. From measured (1)J(NC’) coupling constants in human ubiquitin, characteristic sequences of the coupling constant are observed which correlate with the protein secondary structure. In the alpha-helix, the coupling constant does not vary much (15.3 +/- 0.5 Hz). In beta-sheets, variations are larger (15.5 +/- 1.5 Hz), except in the central region of the parallel and antiparallel P-sheets, where the coupling constant exhibits rather regular values. In this region, significant correlation of the coupling constants within the lines of interstrand N-H (...) O=C hydrogen bonding is observed, indicating a cooperative polarization of peptide bonds in the H-bonding network. The largest change of the coupling constant is found within reverse turns. At position 2 of reverse turns the coupling constant has the lowest values (13.9 +/- 0.8 Hz), whereas at position 4 the values are highest (16.6 +/- 0.6 Hz). This change of the coupling constant within the three residues of reverse turns is caused by specific hydrogen bonding of amide groups in the reverse turns. The result indicates that the intraprotein N-H (...) O=C hydrogen bonds of the main-chain amide groups are weaker than the hydrogen bonds; of these groups to water.