A rapid and simple procedure for the concurrent assay of riboflavin and its coenzyme forms at the picomole level is described. The method makes use of reverse-phase HPLC with fluorometric detection. Some characteristics of the assay of FAD, FMN and riboflavin, respectively, are as follows. Range of linear response, in picomoles: up to 93, 84, 66; mean retention time, in minutes: 3.6, 5.4, 9.4; recovery from standard solutions, in percent (means±S.E.): 92 f 4.7, 84±3.9, 98±0.1. The method has been applied to the determination of flavins in tissue samples such as brain, heart, kidney and liver. The results obtained are comparable to those reported for a conventional fluorometric method.