Hydroxylation of 19-hydroxyandrost-4-ene-3,17-dione (190HA) by aromatase occurs at the 19-pro-R hydrogen, suggesting that the C19 group has a preferred conformation in the enzyme active site. X-ray crystallographic studies have led to a postulate that the steroid plays a role in determining this conformation. In an effort to quantitate the steroid's role, we estimated conformational constraints about the C10-C19 bond of 190HA using molecular mechanics calculations. Rotational barriers ≤6 kcal/mol and energy differences between conformers ≤1 kcal/mol were found. We perturbed these conformational constraints by preparing an altered substrate, 19-hydroxyandrosta-4,6-diene-3,17-dione (190HAD). The stereospecificity of aromatization for 190HA and 190HAD was found to be the same. Thus, theoretical and experimental approaches both indicate that conformational constraints intrinsic to 190HA cannot be a major determinant in the stereospecificity of its oxidation by aromatase.