The folacin-binding characteristics and chromatographic properties of the folacin-binding protein (FBP) of commercial pasteurized skim milk and whey protein concentrate were compared with those of fresh raw cows' milk. Native state FBP recently has been shown to enhance the intestinal absorption of folacin, whereas the FBP of pasteurized milk is ineffective. Anion-exchange chromatography indicated no major electrostatic differences in the FBP of these products, although gel-filtration chromatography provided evidence of enhanced FBP aggregation in the pasteurized whey protein concentrate. Analysis of folic acid binding kinetics by using Scatchard and Hill plots indicated that pasteurization or subsequent processing induces alterations in binding cooperatively, its pH dependence, or both. These results suggest that partial denaturation during pasteurization alters the folacin-binding characteristics and extent of molecular interaction of FBP. These changes may be responsible for the reported differences between raw and pasteurized milk products in their ability to enhance folacin absorption. Further research is needed to clarify the biological significance of these findings with respect to potential differences in folacin bioavailability from breast milk, pasteurized cows' milk and infant formulas.