The interaction of niacin, vitamin B6 and zinc was studied in Wistar rats. Seventy animals were submitted to a four-week depletion period during which they were fed a corn grits diet with no niacin, vitamin B6 or zinc added. These animals also received a 32% ethanol solution. Thirty additional rats were used as controls. After the period of depletion, the deficient animals were divided into five subgroups of 10 animals each. Each group received either the deficient diet, the deficient diet plus niacin, the deficient diet plus vitamin B6, the deficient diet plus zinc, or the control diet for the following two weeks. Five rats from each group were killed weekly after 24-hour urine collection. N'methylnicotinamide (N'MN), N'methyl-2-pyridone-5-carboxamide (2 PYR) and 4-pyridoxic acid (4 PYR) were measured in urine by HPLC. Niacin repletion increased the excretion of niacin metabolites, N'MN and 2 PYR (p less than 0.01), in relation to deficient animals. Niacin and vitamin B6 metabolites increased with vitamin B6 repletion (p less than 0.01). Repletion with zinc alone was followed by an increase in urinary excretion of N'MN (p less than 0.05), 2 PYR (p less than 0.01) and 4 PYR (p less than 0.01). When the deficient rats were fed the control diet containing the three nutrients, all three metabolites increased (p less than 0.01). The main conclusion is that zinc repletion per se caused activation of niacin metabolism, increasing the excretion of niacin metabolites. This emphasizes the role of zinc in the function of these vitamins.