Increased gap junction expression in lamina propria myofibroblasts and urothelial cells may be involved in detrusor overactivity leading to incontinence. Immunohistochemistry was used to compare connexin 26, 43 and 45 expression in the bladders of neonatal, adult and spinal cord transected rats, while optical imaging was used to map the spread of spontaneous activity and the effects of gap junction blockade. Female adult Sprague-Dawley rats were deeply anesthetized, a laminectomy was performed and the spinal cord transected (T8/T9). After 14 days, their bladders and those of age-matched adults (4 month-old) and neonates (7-21 day-old) were excised and studied immunohistochemically using frozen sections or optically using whole bladders stained with voltage- and Ca²⁺-sensitive dyes. The expression of connexin 26 (Cx26) was localized to the urothelium, Cx43 to the lamina propria myofibroblasts and Cx45 to the detrusor smooth muscle. While the expression of Cx45 was comparable in all bladders, the expression of Cx43 and Cx26 was increased in neonate and transected animals. In the bladders of adults, spontaneous activity was initiated at multiple sites resulting in a lack of coordination. Alternatively, in neonate and transected animals spontaneous activity was initiated at a focal site near the dome and spread in a coordinated fashion throughout the bladder. Gap junction blockade (18-glycyrrhetinic acid, 1 µM) abolished this coordinated activity but had no effect on the uncoordinated activity in adult bladders. These data suggest that coordinated spontaneous activity requires gap junction upregulation in urothelial cells and lamina propria myofibroblasts.