Rotational spectra of two conformers of the peptide mimetic, ethyl-acetamidoacetate, were measured in a molecular beam using a Fourier-transform microwave spectrometer. In each conformer, internal rotation of the acetyl methyl group gives rise to observable splittings in the spectrum. From analysis of the torsion-rotation interactions, the methyl group's orientation has been determined in the principal axis frame of each conformer and is shown to unambiguously identify its conformational form. One conformer exists in the all-trans configuration and belongs to C-S point group and the second, higher-energy conformer has C-1 symmetry. Two separate theoretical fitting procedures are applied to assess the reliability of the structural information and are shown to be essentially equivalent. For example, methyl torsional barriers are 63.7(1) cm(-1) versus 67.1(1) cm(-1) and 64.8(1) cm(-1) versus 67.5(1) cm(-1) for the C-S and C-1 conformers, respectively, and principal axis orientations of the methyl groups agree to +/-0.1degrees. The small differences in the torsional barriers and rotor axis angles for the two conformers are a result of a change in the orientation of the ethyl group on the other end of the molecule. The predicted energy ordering of these two conformers at the MP2/6-311G(d,p) level of theory is in disagreement with experimental observations. (C) 2003 American Institute of Physics.