Spontaneous activity is a phenomenon common to many smooth muscle tissues including the gastrointestinal tract, some blood vessels and urinary tract tissues including the bladder. Spontaneous contractility of bladder smooth muscle are non-voiding contractions which are thought to underlie the tone of the bladder wall during filling (Turner and Brading, 1997). While this activity is considered to be myogenic, its origin and modulation is incompletely understood. The aim of the present study was to assess the contribution of the mucosal layer to spontaneous contractility and to examine the effect of pharmacological agents which target gap junctions. Bladders were removed from guinea-pigs, killed by cervical dislocation in accordance with Schedule 1, UK Home Office regulations. In vitro tension recordings were made from bladder strips mounted in organ baths which typically developed spontaneous activity during the equilibration period. Spontaneous activity was unaffected by tetrodotoxin (0.1μM), indicating its myogenic origin (n=19 strips from 8 animals, p>0.05). There was no significant difference in the amplitude or frequency of spontaneous contractions between strips which were intact (7.2±0.8mN, 3.5±2.3 per minute) and those which had the mucosal layer removed (8.1±0.9mN, 2.8±1.7 per minute, p>0.05, n=24 strips from 12 animals, unpaired t-test). Gap 27, a Cx43 gap junction blocker transiently inhibited spontaneous activity in both intact and denuded strips (n=5 from 3 animals). The gap junction uncoupler, heptanol (300μM) completely inhibited activity in intact and denuded strips within 6.13±0.45min and 2.87±0.78 min respectively (p<0.05, n=6 strips from 2 animals, unpaired t-test). Full recovery was obtained on washout. The addition of a gap junction opener, AAP10 did not affect the amplitude or frequency of spontaneous activity in intact or denuded strips (p>0.05, n=3) indicating that gap junctions are fully open under control conditions. Moreover, in intact and denuded strips, the time taken by heptanol to inhibit activity was not significantly affected by pre-treatment with AAP10 (n=5, p>0.05 in each case). The findings of the present study have shown that myogenic spontaneous activity of the bladder requires functional communication via gap junctions and that this activity does not depend on the presence of the mucosal layer.