We investigated the mechanisms underlying the activation of a transient BK current (tBK) in rabbit urethral smooth muscle cells (RUSMCs), using the perforated patch clamp technique at 37°C. The tBK current required an elevation in intracellular Ca²⁺ resulting from RyR activation via Ca²⁺-induced Ca²⁺-release, triggered by Ca²⁺ influx through L-type Ca²⁺ (Ca_V) channels. Carbachol inhibited tBK current by reducing both Ca²⁺ influx and Ca²⁺ release and altered the shape of spike complexes recorded under current clamp. The tBK currents were blocked by iberiotoxin and penitrem A (300 nM and 100 nM, respectively) and were also inhibited when external Ca²⁺ ([Ca²⁺]_o) was removed or the Ca_V channel inhibitors, nifedipine (10 μM) and cadmium (Cd²⁺, 100 μM) were applied. The tBK current was inhibited by caffeine (10 mM), ryanodine (30 μM) and tetracaine (100 μM), suggesting that RyR-mediated Ca²⁺ release contributed to the activation of the tBK current. When IP₃Rs were blocked with 2APB (100 μM) the tBK current was not reduced in amplitude. However, when Ca²⁺ release via IP₃Rs was evoked with phenylephrine (1 μM) or carbachol (1 μM) the tBK current was inhibited. The effect of carbachol was abolished when IP3Rs were blocked with 2APB or by inhibition of muscarinic receptors with the M3 receptor antagonist 4-DAMP (1 μM¬). Under current clamp, bursts of action potentials could be evoked with depolarizing current injection. Carbachol reduced the number and amplitude of spikes in each burst and these effects were reduced in the presence of 2APB.