Rainbow trout (O. mykiss) of 60 g mean weight were given, for 18 weeks, diets containing 0.6, 1.6, 2.7, 5.6 and 12.9 mg riboflavin/kg dry diet. A level of 2.7 mg riboflavin/kg dry diet was required to: (a) prevent the appearance of gross deficiency symptoms including ocular opacities; (b) obtain maximal riboflavin levels in liver and heart; (c) maximal activity of hepatic D-amino acid oxidase (D-AAO); (d) minimal stimulation of the activities of D-AAO, erythrocyte glutathione reductase (EGR) and hepatic glutathione reductase (HGR), after "in vitro" pre-incubation with the coenzyme flavin adenine dinucleotide (FAD). Hepatic D-AAO activity was shown to be a reliable indicator of incipient riboflavin deficiency and EGR a convenient indicator of riboflavin deficiency in the field. © 1990.