In the urinary bladder, the main source of NO seems to be the urothelium and the underlying suburothelium. In this study, we aimed to characterize how receptors in the human urothelium regulate the production and release of NO. For this, we cultured two human urothelial cell lines - the normal immortalized cell line UROtsa and the malignant cell line T24. These were treated with an array of agonists and antagonists with affinity for adrenergic, muscarinic and purinergic receptors. The production of NO and expression of nitric oxide synthase (NOS) was studied by immunocytochemistry and Western blotting. The amount of released NO was measured indirectly by detecting nitrite using amperometry and a Griess reaction kit. The results showed that NO, endothelial NOS and inducible NOS were predominantly produced and expressed in the close vicinity of the nucleus in untreated human urothelial cells. Upon treatment with a beta-adrenoceptor agonist, but not any of the other agonists or antagonists, the pattern of NO production changed, showing a more even production throughout the cytosol. The pattern of expression of endothelial NOS changed in a similar way upon dobutamine treatment. The release of nitrite, as a measurement of NO, increased after treatment with dobutamine from 0.31 ± 0.029 to 1.97 ± 0.18 nmol and 0.80 ± 0.12 to 3.27 ± 0.24 nmol in UROtsa and T24, respectively. In conclusion, our results show that the expression of NOS and production of NO as well as the release of NO from human urothelial cells is regulated by beta-adrenoceptor activation.