Two nitroheterocyclic drugs, nifurtimox (NFX) and benznidazole (BZ), used in the treatment of Chagas' disease have serious side effects attributed to their nitroreduction to reactive metabolites. Here, we report that these drugs reach the mammary tissue and there they could undergo in situ bioactivation. Both were detected in mammary tissue from female Sprague-Dawley rats after their intragastric administration. Only NFX was biotransformed by pure xanthine-oxidoreductase and from tissue cytosol. These activities were purine dependent and were inhibited by allopurinol. Also, only NFX was biotransformed by microsomes in the presence of β-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), and was inhibited by carbon monoxide and partially by diphenyleneiodonium. NFX treatment produced significant decrease in protein sulfhydryl content after 1, 3 and 6 hours; no increases in protein carbonyl content at any time tested and significantly higher levels of lipid hydroperoxides at 3 and 6 hours; besides, ultrastructural observations after 24 hours showed significant differences in epithelial cells compared to control. These findings indicate that NFX might be more deleterious to mammary tissue than BZ and could correlate with early reports on its ability to promote rat mammary tissue toxicity.