The conformation of NADP+ in glucose-6-phosphate-dehydrogenase--NADP+ binary complexes has been investigated using proton-proton transferred nuclear Overhauser enhancement measurements to determine interproton distance ratios between bound NADP+ protons. The enzymes from Saccharomyces cerevisiae (brewer's yeast and baker's yeast) and Hansenula jadinii (Candida utilis, Torula utilis) form binary complexes with NADP+ in which the glycosidic bond of the adenine moiety is in the anti conformation whereas that of the nicotinamide moiety exists as a syn (69-70%)/anti (30-40%) mixture. The enzymes have similar subunit sizes (Mr approximately 58 000) and it is shown that they bind NADP+ in essentially similar conformations. Inactivation of the baker's yeast enzyme with acetylsalicylic acid caused little if any alteration in the conformation of bound NADP+, and the presence of NADP+ during inactivation afforded very little protection to the enzyme. Inactivation rates were, however, lower in the presence of glucose 6-phosphate. It is concluded that the epsilon-amino group of the lysine residue that is acetylated during the inactivation reaction with acetylsalicylic acid is not necessary for binary complex formation between the enzyme and NADP+, but that it is situated in a part of the molecule affected by formation of the enzyme--glucose-6-phosphate complex. The implication of the findings for the catalytic process, and related evolutionary aspects, are discussed briefly.