Increased spontaneous contractile activity in detrusor smooth muscle may contribute to unstable bladder contractions and increased bladder tone associated with urinary incontinence (1). The underlying mechanisms for enhanced contractile function are not clear, but an altered intracellular Ca²⁺ regulation is important (2, 3). The present investigation characterised localised rises of intracellular Ca²⁺ concentration, [Ca²⁺]_i, in detrusor myocytes from guinea-pig and human bladders and examined their role in generating spontaneous activity. Guinea-pigs were humanely killed. Human detrusor samples were obtained from surgical operations, with Ethical Committee approval and patient consent. Single detrusor myocytes were dissociated from muscle strips using a collagenase-based enzyme mixture (4). Cells were superfused with a Tyrode′s solution, gassed with 95% O₂/5% CO₂, at 37°C, pH7.4. Cells were loaded with Fluo-4 AM (1μM) and [Ca²⁺] _i was measured as fluorescence intensity emitted at 515-530 nm, excited at 488 nm, scanned with an argon laser, using a BioRad Radiance 2100 system. Images were obtained either by line scan or x, y plane scan and processed with LaserPix program. Parameters for quantifying Ca²⁺ sparks were amplitude (F/F₀), full duration half-maximum (FDHM) and full-width half-maximum (FWHM). Data are expressed as mean±SD and Student′s t-test was used (p<0.05) Localised rises of [Ca²⁺]_i, in the form of discrete, fast sparks, were observed both in un-stimulated guinea-pig (F/F₀ 1.52±0.33, FDHM 160±89ms, FWHM 2.08±0.85μm, frequency 0.38±0.27Hz, n=89) and human detrusor myocytes (F/F₀ 1.45±0.37, FDHM 142±74ms, FWHM 2.61±0.96μm, frequency 0.27±0.16Hz, n=72). Larger cluster-like local activities also occurred. There was generally one spark-generating site inside the cell, but multiple release sites could also be observed. These localised sparks either remained local or spread to nearby areas. Repetitive firing of Ca²⁺ sparks and their fusion were able to progress into Ca²⁺ waves, leading to synchronised whole cell [Ca²⁺]_i transients. The spark activities could be abolished by 10-20μM ryanodine (n=10), as well as 20μM cyclopiazonic acid (n=6) and 1μM thapsigargin (n=5). A low caffeine concentration (1mM) and a moderate rise of extracellular KCl (to 20mM) increased spark frequencies (caffeine: 162±14% of control, p<0.01 n=4; KCl: 140±25% of control, p<0.05, n=3) and led to subsequent whole cell transients. These observations show that localised Ca²⁺ rises can occur spontaneously in detrusor smooth muscle cells at rest. The temporal and spatial summation of these local Ca²⁺ oscillating events leads to spontaneous global activity in detrusor muscle. These local transients are controlled by the activity of ryanodine receptors, and can be up-regulated by membrane depolarisation.