The "azido gauche effect" was examined both experimentally and theoretically and was found to determine the conformation of, for example, (4R)-and (4S)-azidoproline (Azp) derivatives. For (4R) Azp derivatives, the azido gauche effect induces a preferred C(4)-exo conformation of the pyrrolidine ring, which leads to stabilization of the s-trans amide conformer of, e.g., Ac-(4R) Azp-OCH3 (5R) via an n ->pi* interaction between the nonbonding electrons of the oxygen of the acetyl group and the carbonyl group of the ester. For (4S) Azp derivatives, the azido gauche effect results in a C(4)-endo conformation of the pyrrolidine ring that does not allow for this stabilizing n ->pi* interaction of the s-trans conformer. Consequently, a significantly higher s-trans: s-cis amide conformer ratio is observed for (4R) Azp compared to (4S) Azp derivatives (e. g., 6.1:1 versus 2.6:1 in D2O for Ac-(4R) Azp-OCH3 (5R) compared to Ac-(4S) Azp-OCH3 (5S)). These conformational preferences are reflected in the higher tendency of (4S) Azp-containing peptides to form cyclic peptides with all-cis amide bonds compared to (4R) Azp derivatives. Ab initio calculations demonstrate that the strength of the azido gauche effect is comparable to that of the well-known "fluorine gauche effect". For azidoethane derivatives N-3-CH2CH2-X (X = N-3, NHCOH, NHAc, or N(CH3)Ac), the ab initio calculations revealed energy differences of 5-13 kJ mol(-1) between the anti and gauche conformations in favor of the gauche conformer. Calculations were also performed for the (4R) Azp and (4S) Azp derivatives 5R and 5S, supporting the experimentally observed data.