The inherent conformation of the biotin bicyclic moiety has been examined. In biotin and various heterobiotins the endo conformation of the bicyclic ring is always observed; however, the presence of a side chain in the native coenzyme and its analogs may preclude the alternative exo conformation, masking what may be an inherently more favored conformation. The crystal structures of the three chainless analogs of biotin, oxybiotin, and azabiotin have been determined, and in all cases the bicyclic rings adopt the endo conformation, with the heteroatom proximal to the ureido ring. In contrast, ah initio quantum calculations and high-resolution nuclear magnetic resonance experiments on the chainless analog of biotin suggest that the endo and exo conformations of the bicyclic ring are of comparable energy and coexist in approximately equal amounts in solution.