The purpose of the present work was to examine effects of the Cu–Fe interaction on intestinal β-carotene 15,15′-dioxygenase activity when a wide range of dietary Fe (deficiency to excess) was used in relation to Cu status of rats. The effect of dietary carbohydrates was also examined since they play a role in the Cu–Fe interaction in vivo. Weanling male Sprague-Dawley rats (n 72) were divided into twelve dietary groups, which were fed on either low-, normal-, or high-Fe levels (0·9, 9·0, and 90·0 mmol Fe/kg diet respectively) combined with Cu-adequate or -deficient levels (0·94 and 0·09 mmol Cu/kg diet respectively) and with starch or fructose in the diets. The data showed that both Fe concentration and β-carotene 15,15′-dioxygenase activity in small intestinal mucosa were enhanced with increasing dietary Fe and with Cu deficiency v. Cu adequacy. Dietary fructose did not aggravate the Fe-enhancement, related to Cu deficiency, in the small intestine; however, fructose increased the intestinal dioxygenase activity in rats fed on normal- or high-Fe diets when compared with starch controls. Thus, the highest intestinal dioxygenase activity associated with the lowest hepatic retinol (total) concentration was found in rats fed on the Cu-deficient, high-Fe, fructose-based diet. Finally, a positive linear relationship was found between the dioxygenase activity and Fe concentration in intestinal mucosa. In conclusion, the data indicate that β-carotene 15,15′-dioxygenase activity requires Fe as cofactor in vivoand the enzyme is modulated by the three dietary components: Cu, Fe, and fructose.