The effect of the heat treatment of casein in presence of reducing sugars on some aspects of Zn availability was investigated. Samples were prepared by mixing casein with glucose-fructose, and were used unprocessed (C) or heated (HC). Changes in Zn speciation after the in vitro digestion of the samples, both as part of a diet and in isolation, were studied. The uptake of soluble Zn from the digested samples was investigated in Caco-2 cells. After in vitro digestion, the percentage of precipitated Zn was significantly higher with the HC sample, both when digested alone and as a part of the diet. In assays with Caco-2 cells, a significant decrease in Zn uptake was observed when the uptake buffer contained the sample C digest, by comparison with the control buffer, without casein digest. When the digested heated mixture was added, Zn uptake by the cells was significantly lower than in either of the two other cases. It may be concluded that the heat treatment of casein in the presence of glucose-fructose has a negative effect on Zn availability because, after in vitro digestion, Zn insolubilization was enhanced and Zn uptake by the enterocyte was impaired, compared with the unheated mixture. In addition, the usefulness of Caco-2 cells in this kind of research has been shown.