Intravesical treatment with cytotoxic drugs such as doxorubicin is associated with local adverse effects in bladder cancer patients. Here we investigate the effects of doxorubicin on urothelial release of ATP, acetylcholine and prostaglandin E2, and production of inflammatory cytokines. Urothelial cells were treated with doxorubicin for 1 h at 37 °C. Immediately or 24 h following treatment the level of ATP, acetylcholine and prostaglandin E2 released under basal and stimulated conditions was measured and compared to release from vehicle treated control cultures. The presence of inflammatory cytokines, in culture medium was also assessed 24 h after doxorubicin pre-treatment. Immediately following treatment, stimulated ATP release was inhibited at doxorubicin concentrations ≥1 μg/ml and showed partial recovery at 24 h. Immediately following treatment, basal acetylcholine release was increased by doxorubicin at its clinical concentration (1 mg/ml), while a concentration-dependent decrease in stimulated acetylcholine release was observed. Twenty four hour after treatment, basal acetylcholine release was increased in culture treated with 0.01 mg/ml doxorubicin while stimulated acetylcholine release remained depressed. A significant increase in prostaglandin E2 release was observed in cells immediately and 24 h after treatment with doxorubicin. A 5.5- and 2-fold increase in interleukin -8 and -1β secretion, respectively was detected 24 h following doxorubicin treatment. These findings indicate that inflammatory cytokines interleukin-8 and -1β are induced and urothelial mediator release is affected by treatment with doxorubicin at clinically relevant concentrations and durations of treatment. These changes may play a role in the adverse effects associated with intravesical doxorubicin treatment.