Adult tissue stem cells replicate infrequently, retaining DNA nucleotide label (BrdU) for much longer periods than mature, dividing cells in which the label is diluted during a chase period. Those label retaining cells (LRCs) have been identified as the tissue stem cells in skin, cornea, intestine and prostate. However, in the urinary tract uroepithelial stem cells have not yet been identified. In this study, BrdU administration identified urothelial LRCs in the rat bladder with nine percent of the epithelial basal cells retaining BrdU label one year after its administration. Markers for stem cells in other tissues, Bcl, p63, cytokeratin 14 and 1 integrin, were immunolocalized in the basal bladder epithelium in or near urothelial LRCs, but not uniquely limited to these cells. Flow cytometry demonstrated that urothelial LRCs were small, had low granularity and were uniquely 4 integrin-bright. Urothelium from long-term labeled bladders was cultured and LRCs were found to be significantly more clonogenic and proliferative, characteristics of stem cells, than unlabeled urothelial cells. Thus, this work demonstrates that LRCs in the bladder localize to the basal layer, are small, low granularity, uniquely β4 integrin-rich, slowly-cycling and demonstrate superior clonogenic and proliferative ability compared to unlabeled epithelial cells. We propose that LRCs represent putative urothelial stem cells.