Abstract The binding and function of FMN, 3,6,7-trimethyl-9-(1'-d-ribityl)isoalloxazine 5'-phosphate (3-methyl-FMN),6,7-dichloro-9-(1'-d-ribityl)isoalloxazine 5'-phosphate (6,7-dichloro-FMN), and 6,7-dimethyl-9-(5'-hydroxypentyl)isoalloxazine 5'-phosphate (5'-hydroxypentyl-FMN) has been investigated with reduced nicotinamide adenine dinucleotide phosphate dehydrogenase and NADPH cytochrome c reductase from yeast, NADH oxidase from a pseudomonad, pyridoxamine phosphate oxidase from liver, and glycolate oxidase from spinach. Considerable variation has been observed in the quantitative behavior of the flavin phosphates with the differing apoenzymes through measurements of Vmax and Km or Ki. The earlier postulation that the 3-imino group of the isoalloxazine is primary point of attachment of the flavin phosphate to an FMN-dependent apoenzyme must now be modified, especially for the NADPH dehydrogenase (old yellow enzyme) which has been considered to be the prototype of flavin coenzyme-apoenzyme interaction. Present observations with 3-methyl-FMN indicate a lack of absolute dependence on the 3-imino group and suggest a rather diffuse interaction through mutual polarization or partial charge transfer between relatively large portions of the isoalloxazine structure and perhaps aromatic amino acid residues in the proteins. The generally better binding than function of 6,7-dichloro-FMN indicates that the relatively poor function of similar analogues may be expected with substituent changes in position 6 and 7 which are of the approximate size of methyl groups but through contribution to the electromeric structure of the isoalloxazine markedly change the oxidation-reduction potential. The optimal requirement for a full complement of secondary hydroxyl groups in d configuration on the side chain in position 9 is apparent from the generally decreased coenzymatic activity seen with 5'-hydroxypentyl-FMN. However, there is no absolute dependence on interaction of the enzymes with each secondary hydroxyl group of a flavin phosphate, but rather a steric relationship which favors proper disposition of coenzyme to apoenzyme.