Abstract A competitive binding procedure that can be used to determine either riboflavin or riboflavin-binding protein has been developed. Riboflavin-binding protein from chicken egg white binds tightly to DEAE-cellulose while free riboflavin does not. Stock [2-14C]riboflavin solutions, diluted with varying amounts of a standard unlabeled riboflavin solution or an unknown sample, are mixed with aporiboflavin-binding protein and washed through small DEAE-cellulose columns. The protein-bound riboflavin is batch eluted into scintillation vials, counted, and the unknown samples compared to a standard curve. This is a simple, rapid method for assaying riboflavin by isotope dilution. By a slight modification of the incubation conditions of this procedure, the degree of saturation and amount of riboflavin-binding protein can be determined. Data from both assays can be represented by linear plots in which slopes or intercepts correspond to unknown values. The principles presented here have been extended to the assay of biotin and avidin and should apply to other vitamins and vitamin-binding proteins.