The electrophoretic distribution and substrate specificities of isozymes of glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) were studied in seven species of teleost fish. The fish examined included two species of bonefish, Albula neoguinaica and A. glossodonta (Albulidae, Anquilliformes) (Shaklee and Tamaru, '811, and five representatives of the order Perciformes: two species of butterflyfish, Chaetodon miliaris and C. aurega (Chaetodontidae); a goatfish, Upeneus urge (Mullidae); a goby, Bathygobius fuscus (Gobiidae); and a snapper, Pristipomoides filamentosus (Lutjanidae). After horizontal starch gel electrophoresis, gel slices were stained using a variety of substrates and cofactors. In all species except the goby, two groups of isozymes were distinguished, corresponding to the mammalian G6PD (specific for glucose-6-phosphate (G6P) and NADP') and H6PD (capable of utilizing galactose-6-phosphate and in certain cases other monosaccharide phosphates in addition to GGP). None of the five visible isozymes in the goby was specific for G6P. In each of the other species a single G6P-and NADP+specific isozyme was noted, having the most rapid mobility toward the anode. In addition, it was found that all of the isozymes in all of the fish examined could catalyze the oxidation of fructose-6-phosphate at a rate comparable to that for G6P, suggesting that glucose-6-phosphate dehydrogenase can obviate the role of glucosephosphate isomerase in monosaccharide metabolism.