A role for metallothionein in intestinal zinc absorption has been the subject of considerable debate. If metallothionein affects zinc absorption, then those factors that induce metallothionein synthesis (e.g., heavy metals, hormones) should alter zinc absorption and homeostasis. The present studies used metallothionein transgenic mice (overexpressing) and metallothionein knockout mice (no expression of metallothionein-1 or metallothionein-2) to examine directly the effects of metallothionein on zinc absorption, independent of secondary effects that could be caused by metallothionein inducers. Zinc absorption was examined by administering a single oral zinc dose (0.5 mmol/kg) by feeding tube to metallothionein transgenic and metallothionein knockout mice and measuring the serum zinc concentration. Two hours after the dose, the serum zinc concentration was 2.3 times higher in metallothionein knockout mice than in their control strain. Conversely, the concentration was elevated only one third as much in the metallothionein transgenic mice as in their controls after the zinc dose. We found that the serum zinc concentration was inversely related to the level of metallothionein protein. The intestinal zinc content was higher in the metallothionein knockout mice, however, suggesting that metallothionein did not reduce zinc absorption by simply sequestering zinc in the mucosa. The expression of the zinc transporter ZnT-1 was directly related to the serum zinc level and was independent of the level of metallothionein. These results further support metallothionein as an important component for reducing the efficiency of zinc absorption at elevated zinc intakes.