A NMR and molecular mechanics analysis of isomeric N- and C-terrninal glycyl dipeptides in aqueous solutions is described, with the overall objective of understanding the basis of their different rotameric preferences. The C-terminal glycyl dipeptides are signhicantly more conformationally homogeneous than their N-terminal isomers in all water-containing solvents, as probed by the magnitude of the chemical shift non-equivalence of the diastereotopic glycyl methylene protons in the 1H NMR spectra of both isomers. Further NMR experiments demonstrated that, with both sets of isomers, the degree of conformational homogeneity is under control of the extent of intramolecular ion pairing; the ion pairing is thus a greater conformational determinant in C-terminal glycyl dipeptides. Molecular mechanics studies failed to demonstrate an inherent structural basis for the differences in rotameric preferences between isomers. To explain the differences, it is hypothesized that alkyl substitution of the α-carbon in C-terminal glycyl dipeptides destabilizes the ammonium ion to a greater extent through steric inhibition of solvation than in N-terminal dipeptides, which forces intramolecular ion pairing to occur to a greater extent. The significance of these results towards the de novo prediction of polypeptide tertiary structure in solution is discussed briefly.