1. Bladders were excised from deeply anesthetized (urethane, 1.2 gm • kg−1, i.p.) Sprague Dawley rats (of either sex), cut open, and gently stretched (urothelial side down).
2. Anesthesia was determined to be adequate for surgery by periodically testing for the absence of a withdrawal reflex to a strong pinch of a hind paw and absence of an eye-blink reflex to tactile stimulation of the cornea.
3. After tissue removal, all animals were killed via overdose of anesthetic.
4. The tissue was incubated overnight in primary minimal essential medium, penicillin/streptomycin/fungizone, and 2.5 mg • ml−1 dispase.
5. The urothelium was gently scraped from underlying tissue, treated with 0.25% trypsin, and resuspended in primary cell culture medium.
6. The dissociated cell suspension (0.1 ml, 50,000–150,000 cells ml−1) was plated on the surface of collagen-coated dishes and maintained in culture for 1–3 d.
7. Because long-term maintenance of cells in culture could significantly change the properties of some types of cells, the cells in this study were examined after a short time in culture.
8. In general, all cells were used within the first 3 d after plating.
9. All cells in these cultures were cytokeratin positive and therefore were presumably of epithelial origin.

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