The bioavailability of copper in wheat flour is reported based on chemical fractionation using water, diethyl ether, n-hexane, methanol, acetone, and chloroform/methanol to identify copper-bound matrix sites. The total copper concentrations in the fractions were determined by inductively coupled plasma-mass spectrometry. In vitro gastrointestinal digestion was performed using pepsin and pancreatin enzyme solutions at defined pH values for bioavailability studies. The bioavailable forms of copper in the samples were 61-80% of the total concentration. Extraction with n-octanol and activated carbon adsorption studies were evaluated to compare their efficiencies to the enzymatic approach.