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The UROtsa cell line is a unique model of human urothelium with considerable utility in toxicology-based research. UROtsa cells maintained in serum-free medium have several morphological features of transitional epithelium of the urinary bladder including a multilayered appearance and apically located umbrella-like cells that exhibit tight junctions. These cells also have a basal expression pattern of heat shock protein (hsp) and metallothionein (MT) genes similar to that observed in in situ urothelium. To characterize the functional heat shock and metallothionein stress response in UROtsa cells, confluent cultures grown in serum-free medium were treated with a variety of acute and chronic stresses. These included short-term (1–4 hours) exposures to heat (42.5°C), NaAsO2 (100 μM) or CdCl 2 (53.4 μM) as well as long-term (16 days) treatments with NaAsO 2 (1, 4 and 8 μM) or CdCl2 (1, 5 and 9 μM). No change in hsp 27 gene or protein expression was observed with any of the treatments, but hsp 60, hsp 70A, hsp 70B and hsp 70C mRNAs and hsp 70 protein were induced by the three acute treatments. In terms of the heat shock response, UROtsa cells responded to acute stress in a manner seemingly independent of the stressor, but continuous exposure to NaAsO2 and CdCl2 demonstrated differential gene and protein expression between the treatments, particularly with the highest concentrations of NaAsO2 (8 μM) and CdCl 2 (9 μM). Metallothionein gene and protein expression were also examined in the UROtsa cells treated with cadmium, and increased expression of MT-1E, MT-1X, MT-2A and MT-4 were observed in both the acute and chronic treatments. MT-3 was induced in the cells treated with 53.4 μM CdCl2 for 4 hours, but not in the cultures treated with 1, 5 or 9 μM CdCl2. The morphological similarities shared between UROtsa cells and transitional urothelium of the human urinary bladder combined with a functional stress response characterize the UROtsa as valuable model for studying bladder toxicology.