Interstitial cystitis (IC) is an idiopathic hypersensory condition of the bladder associated with increased urinary adenosine triphosphate (ATP) and increased stretch-activated ATP release by bladder urothelial cells (BUC) suggesting augmented purinergic signaling in the bladder. To test this theory further, monolayers of cultured bladder urothelial cells (BUC) derived from bladder biopsies obtained from IC and normal (control) patients were stimulated with 10 - 30 µM ATP with subsequent measurements of levels of extracellular ATP using the luciferin-luciferase assay. Stimulation with 30 µM ATP resulted in IC supernatant containing several fold more ATP than control BUC initially, followed by a slower subsequent decrease in ATP levels. This difference in ATP levels was not completely due to activity of cellular ecto-ATPase because blockade with ARL67156 did not normalize the difference. Exposure to hypotonic solutions resulted in similar extracellular ATP concentrations in IC and normal BUC, but there was a slower decrease of ATP levels in IC supernatants. Treatment of IC BUC with 10 - 30 µM suramin, a non-specific P2 receptor antagonist, significantly attenuated IC BUC's response to extracellular ATP restoring IC BUC to a normal phenotype. Pre-treatment of IC BUC with 20 ng/mL of heparin-binding epidermal growth factor-like growth factor (HB-EGF), which has been previously been shown to be decreased in IC urine specimens, also restored IC BUC to a normal phenotype with respect to response to ATP stimulation. In conclusion, IC BUC have augmented extracellular ATP signaling which could be blocked by suramin and HB-EGF. These findings suggest future novel therapeutic avenues.