Trypsinization (WT) was employed to disaggregate urothelial cells from normal human urinary bladder mucosa for the preparation of primary cultures. Urinary bladders of two male adults both 25 years old were obtained autopsy 1-2 h after death. The mucosa was incubated in HBSS containing 0.25% trypsin at 37 degrees C. Mean cell yield, viability, and attachment were 14.6 X 10(7), 76%, and 42.5% after WT. In histologic sections of treated mucosa, most of the urothelium was removed by WT. Following plating and attachment, cells obtained by WT formed a monolayer of flattened epithelial-like cells. When stained with polyclonal antikeratin antibody using the indirect immunoperoxidase technique, all of the cells were immunoreactive indicating an epithelial origin. In conclusion, based on morphology and immunocytochemistry, WT removed virtually all of the urothelial cells from the mucosa and no contamination by mesenchymal cells resulted from this procedure. Thus, WT is an appropriate technique for the isolation of urothelial cells and initiation of primary cultures of normal human urothelial cells for subsequent study.