The conformation of a novel 1,2,5-trisubstituted hexahydro-3-oxa-1H-1, 4-diazepine system (DAP) has been investigated by proton and carbon NMR and refined with computer simulations. Four N-1-acetyl-5-(N,N-dimethylamino)carbonyl DAP analogues, differing in the chirality and substituents at C2, have been studied 2-benzyl-(2S,5S)-DAP (SS-F), 2-benzyl-(2R,5S)-DAP (RS-F), 2-methyl-(ZS,5S)-DAP (SS-A), and 2-methyl-(2R,5S)-DAP (RSA). The NMR spectra for each of the four analogues showed the presence of two configurational isomers slowly interconverting on the NMR time scale; the site of the cis/trans isomerization was identified as the N1-acetyl peptidic bond. The experimental restraints from NMR data consisted of a limited number of interproton distances and well-determined coupling constants; their utilization in distance geometry and distance and angle driven dynamics calculations produced high-resolution structures for three of the analogues. The C2 chirality (R or S) in the seven-membered ring determines the topological projection of the acetyl group with respect to the plane of the ring. The nature of the C2 substituent has only a small effect on the conformation of the ring and overall orientation of the Nt and C5 substituents. No significant structural differences result from the cis or trans configuration at the N1-acetyl peptidic bond. The results presented here indicate that DAP is a useful dipeptidomimetic with structural characteristics distinct from those of the widely used benzodiazepine.