Prostaglandins (PG), particularly PGE₂, are implicated in the development of bladder overactivity. Although PGE₂ causes contraction of the mouse bladder, its mechanism of action has not yet been fully elucidated. This study used contraction studies and a confocal Ca²⁺-imaging technique to further investigate the mechanism of action of PGE₂. Female CD-1 mice were killed by rising CO₂ and cervical dislocation; the bladder removed and strips were cut (from trigone to vertex) with urothelium left intact. <p>Exogenously applied PGE₂ (50 μM) caused an initial slow contraction of mouse bladder strips (peaking at around 2.23 ± 0.17 mN, number of animals, n = 7) and induced subsequent spontaneous contractions (with a mean frequency of 0.21 ± 0.01 Hz, and amplitude of 0.41 ± 0.05 mN, n = 7). Such activity in vitro is a characteristic of bladder overactivity. The PGE₂-induced overactivity was blocked by the L-type Ca²⁺ channel antagonist, nifedipine (1 μM) (P < 0.01, n = 6). Recent work from our lab has shown that spontaneous electrical activity at rest (in mouse urinary bladder) is neurogenic in origin; spontaneous action potentials result from stochastic ATP release from parasympathetic nerves (1), however the PGE₂-induced spontaneous contractions seen in vitro were unaffected by the selective P2X₁ antagonist NF449 (10 μM) (P = NS, n = 4). <p>A confocal Ca²⁺-imaging technique was used to identify Ca²⁺ changes in bladder smooth muscle cells loaded with the Ca²⁺-indicator, Oregon Green 488 BAPTA-1 AM (10 μM). Bath application of PGE₂ (50 μM) significantly increased the frequency of spontaneous whole cell Ca²⁺ flashes (from 0.04 ± 0.01 Hz to 0.18 ± 0.02 Hz, P < 0.01, n = 11), but had no effect on the amplitude of these flashes (137 ± 32%, as a percentage of control, P = NS, n = 5). The PGE₂-induced increase in the frequency of flashes was abolished by nifedipine (1 μM) (P < 0.01, n = 4), but were not significantly reduced in frequency by NF449 (10 μM) (83 ± 15%, P = NS, n = 4). In addition, the synchronicity of PGE₂ induced whole cell Ca²⁺ flashes observed may indicate that an increased level of coupling occurs in smooth muscle cells in response to the application of PGE₂. <p>These data suggest that PGE₂ directly or indirectly activates L-type Ca²⁺ channels (Ca_V1 family) to induce spontaneous contractions in bladder smooth muscle cells and that the purinergic P2X₁ receptor is not significantly involved in such a response.