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-OCH(3) (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 D(2)O for Ac-(4R)Azp-OCH(3) (5R) compared to Ac-(4S)Azp-OCH(3) (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)-CH(2)CH(2)-X (X = N(3), NHCOH, NHAc, or N(CH(3))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.