Previous studies have concluded that copper might inhibit enamel demineralization in vitro. Our aim was to assess the effect of copper (Cu2+), with and without amine fluoride, on human dental enamel under cariogenic challenge in situ. In a double-blind randomized four-leg crossover trial, 14 individuals wore a removable appliance containing 2 enamel slabs, 1 containing an artificial caries lesion. During each leg, the appliance was exposed twice daily to one of the test solutions: 1.25 mM CuSO4, amine fluoride (250 ppm F), copper and amine fluoride combined, or a placebo (water). A cariogenic challenge was provided in all cases by 5 daily exposures to 10% sucrose. Slabs were assessed before and after 21 days' exposure by Knoop microhardness and transverse microradiography. Significantly less demineralization was observed with Cu2+ and fluoride in combination than with fluoride treatment alone (p < 0.05), whereas copper alone had no significant protective effect.