The effects of alkaline pH and elevated sodium concentrations in culture medium on rat bladder explants for 1, 2, and 3 weeks were investigated by continuous BrdU labeling and histopathology. Increasing the sodium chloride concentration of normal medium by 50 or 100 mM caused slight urothelial hyperplasia with statistically significant increases in labeling in week 2 (50 mM) and at all time points with 100 mM NaCl. Cytotoxicity was seen in the high salt group. Increasing the pH from 7.2 to 7.8 and 8.2 also caused a slight hyperplastic response with significant increases in labeling and cytotoxicity at pH 8.2. However, bladder explants treated at pH 7.8 or 8.2 with excess sodium concentrations of 50 to 100 mM had a more marked hyperplastic response with evidence of cytotoxicity as well. There were significant increases in the labeling index (6.4- to 15.0-fold relative to control) after 1 week, with the maximum response at 100 mM sodium/pH 8.2. These results suggest that alkaline pH and elevated sodium concentration have a direct mitogenic effect on rat urothelial cells with some cytotoxicity-induced regenerative cell proliferation as well. These in vitro results in an organ culture system are in agreement with in vivo studies that have shown an important role for elevated urinary cation concentrations and pH in the stimulation of DNA synthesis, induction of hyperplasia, and tumor promotion in rat bladder epithelium.