SummaryAn assay method involving the use of radioactive biotin and Sephadex chromatography was used to determine avidin and/or the avidin-biotin complex. It was demonstrated that avidin is relatively heat labile while the avidin-biotin complex is relatively heat stable. It was found that the affinity of avidin for biotin, essentially zero in aqueous solution, is a direct function of the ionic strength of the medium in which avidin and biotin react.