The role of ascorbic acid in scavenging free radicals was evaluated in a model of mammalian colonic epithelium homogenized in physiologic buffer and exposed to ionizing radiation. Ascorbic acid interacts with hydroxyl free radicals, resulting in production of the ascorbate free radical (AFR). Colonic mucosa contains a soluble factor that is heat sensitive, PCA precipitable and is contained within 1,000 MW dialysis tubing; it uses GSH and cysteine to reduce AFR. The factor from rat colon is fractionated between 55 and 70% saturation with solid (NH4)2SO4; a 3-4 fold increase in enzyme activity was achieved. We suggest that the factor is a cytosolic enzyme appropriately referred to as soluble AFR-reductase. This information provides insight into the mechanism by which ascorbic acid protects against damage by hydroxyl free radicals.