In recent years it has become clear that lipid microdomains within cellular membranes are important for cell signalling processes. Membrane lipid rafts, i.e. regions enriched in cholesterol and sphingomyelin, have been identified in many cell types including smooth muscle. Depletion of cholesterol using either methyl-β-cyclodextrin (MCD) or cholesterol oxidase causes substantial changes in phasic contractions of ureteric smooth muscle (Babiychuk et al., 2004). In the present study we analysed mechanism of stimulatory action of cholesterol extraction in freshly isolated cells. Late pregnant (19-21 days) Wistar rats were killed by cervical dislocation after CO₂ anaesthesia, in accordance with Schedule 1 of the UK Home Office. Single cells were enzymatically isolated from the longitudinal layer of the myometrium and superfused with Krebs solution. Conventional whole-cell patch clamp technique was used to measure transmembrane currents. Spontaneous and oxytocin-induced [Ca²⁺]_i transients were recorded from cells loaded with Fluo-4/AM (7mM, 10 min at 35°C) using confocal microscope (Perkin Elmer UltraView™ LCI). The amplitude of [Ca²⁺]_i transients was expressed as normalised Fluo-4 fluorescence (ΔDF/F0). Records were made from the same cell before and after cholesterol extraction, which was achieved by 10 minutes incubation of cells in Krebs solution containing 2% MCD followed by 5 min washout. In spontaneously active cells extraction of cholesterol significantly increased the frequency of [Ca²⁺]_i transients from 0.34±0.03 to 1.46±0.11 Hz and their amplitude from 0.25±0.01 to 0.8±0.03 (t-test , p<0.01, n=36, data expressed as mean ± standard error). Application of 10nM oxytocin to quiescent cells caused a bi-phasic increase in [Ca²⁺]_i due to (i) release of Ca²⁺ from the SR and (ii) initiation of action potentials. The amplitude of the oxytocin-induced SR Ca²⁺ release was not affected by extraction of cholesterol (paired t-test, 0.53±0.15 in control vs. 0.51±0.14, p=0.93, n=7). Parameters of the [Ca²⁺]_i transients evoked by oxytocin-induced action potentials were increased by cholesterol extraction similarly to that of spontaneous [Ca²⁺]_i transients. In voltage clamped cells, cholesterol extraction led to a decrease in the potassium current density from 25.5±0.3 pA/pF to 17.1±0.12 pA/pF and increase in non-selective conductance (from 0.76±0.07 to 1.67±0.12nS) without a change in cell capacitance. These data suggest a stimulatory action of cholesterol extraction on uterine myocytes, mediated by reduction in outward K⁺ current and increase in inward current possibly due to activation of non-selective cationic conductance.