Diuron, a substituted urea herbicide, is carcinogenic to the urinary bladder of rats at high dietary levels. Its proposed carcinogenic mode of action (MOA) includes urothelial cytotoxicity and necrosis followed by regenerative cell proliferation and sustained urothelial hyperplasia. Cytotoxicity could be induced either by urinary solids or by chemical toxicity by diuron and/or metabolites excreted in the urine. Diuron was not genotoxic in a previous single-cell gel (comet) assay, but possible cross-linking activity remained to be evaluated. The present study explored the MOA of diuron and the effect of urinary acidification on the development of urothelial lesions. Male Wistar rats were fed diuron (2500 ppm, about 130 mg/kg of body weight) either with or without NH(4)Cl 10,000 ppm to acidify the urine. Reversibility of urothelial changes was also examined. The animals were euthanized after 15, 25, or 30 weeks. Diuron-fed rats had urinary amorphous precipitate and magnesium ammonium phosphate crystals similar to control animals. Groups treated with diuron + NH(4)Cl showed decreased urinary pH and reduced amounts of urinary crystals and precipitate. Urothelial necrosis and simple hyperplasia were observed by light microscopy and scanning electron microscopy both in diuron- and in diuron + NH(4)Cl-treated groups. Cytotoxicity and proliferative changes were mostly reversible. A modified comet assay developed in vitro with Chinese hamster ovary cells showed that diuron did not induce DNA cross-links. These data suggest that cytotoxicity with consequent regenerative cell proliferation is the predominant MOA for diuron rat urothelial carcinogenesis, the cytotoxicity being chemically induced and not due to urinary solids.