Within the structure determination task for peptides, which is of large interest due to the relation between structure and functionality, infrared spectra can provide detailed information on the conformational behavior. The conformational landscape of N-acetylgycine has been studied by a combined theoretical and matrix-isolation FT-IR study. The acetylation simulates an amino acid a peptide bond. Four stable conformations were found at the MP2/6-31++G** level of theory. Among these, only one contains an intramolecular H-bond that has a small abundance at the considered temperature. Apart from this one, three other different conformations could be detected in an Ar matrix. The experimental rotamerization constants NAG2 (sic) NAG1 and NAG3 (sic) NAG1 could be estimated. The values of the rotamerization constants as well as the mean frequency deviation of N-acetylglycine were combined with previously obtained data of other N-acetylated amino acids and they appeared to be similar to the data for nonsubstituted amino acids. This suggests that the used methodology can be in the future applied to investigate small peptides. Analysis of H-bond frequency shifts and distance demonstrates that the intramolecular H-bonds in N-acetylated amino acids are stronger compared to those in nonsubstituted amino acids.