The conformation of an uncomplexed form of the antibiotic valinomycin (C(54)N(6)O(18)H(90)) has been determined by direct methods including a novel technique for strong enantiomorph discrimination via the calculation and systematic analysis of cosine invariants of a special type. The intramolecular hydrogen bonding scheme and the isopropyl group stereochemistry of uncomplexed valinomycin are compatible with interpretations of spectral measurements for the complexed and uncomplexed molecule in solution but are different from any previously proposed structure. The simple conformational change of a hydrogen bond shift, which could be induced by the process of potassium ion complexing, transforms the uncomplexed into the complexed structure.