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The experiments reported here were carried out to study the in vitro binding of 131 I to bovine milk protein and the factors influencing this binding. Incubation of inorganic carrier-free 131 I-iodide with fresh raw milk at 37C for two hours showed as much as 50% of the 131 I bound to milk protein, and that as much as 80–90% could be bound in 12–24 hours. It was also shown that this reaction is both time- and temperature-dependent and could be readily blocked by incubation at 8C, boiling the milk prior to incubation, addition of carrier iodide or antithyroid agents such as Tapazole (methyl mercaptan imidazole). Paper chromatography of enzymatic digests of the milk showed the protein-bound 131 I in milk is present as 131 I-labeled monoiodotyrosine. No other 131 I-labeled compounds were observed. These studies would tend to indicate that the binding of 131 I-iodide to milk protein involves an enzymatic oxidation of the iodide.