The effects of pseudomonal virulence factor pyocyanin, and LPS from Pseudomonas aeruginosa and Escherichia coli on urothelial mediator release and cytokine production were examined. RT4 urothelial cells were treated with pyocyanin (1-100μM) or LPS (1-100ng/mL) for 24-h. Effects were measured in terms of changes in cell viability, basal and stretch-induced acetylcholine (Ach) and PGE2 release, and inflammatory cytokines (IL-6 and IL-12) production. Twenty-four hour pyocyanin (100μM) treatment significantly decreased urothelial cell viability, while stretch-induced Ach release response was inhibited. E. coli LPS (100ng/mL) produced a similar response with an additional significant increase in basal Ach release. All three virulence factors significantly increased urothelial PGE2 release; under basal release for pyocyanin (100μM), stretch-induced release for pseudomonal LPS (≥10ng/mL) and both basal and stimulated release for E. coli LPS (≥10ng/mL). IL-6 and IL-12 were not detected in control samples, however 24h treatment with pyocyanin (100μM) or LPS (100ng/mL) resulted in IL-6 release from urothelial cells. The changes in urothelial Ach and PGE2, and release of inflammatory cytokine IL-6 induced by exposure to the bacterial virulence factors may play a role in the symptoms of pain and urinary urgency experienced with urinary tract infections.