Stimulation of -adrenoceptors contributes to the relaxation of urinary bladder smooth muscle (UBSM) through activation of large conductance Ca²⁺-activated K⁺ (BK) channels. We examined the mechanisms by which -adrenoceptor stimulation leads to an elevation of the activity of BK channels in UBSM. Depolarization from -70 to +10 mV evokes an inward L-type voltage dependent Ca²⁺ channel (VDCC) current followed by outward steady-state and transient BK current. In the presence of ryanodine, which blocks the transient BK currents, isoproterenol, a non-selective -adrenoceptor agonist, increased the VDCC current by approximately 25% and the steady-state BK current by about 30%. In the presence of the BK channel inhibitor iberiotoxin, isoproterenol did not cause activation of the remaining steady-state K⁺ current component. Decreasing Ca²⁺ influx through VDCC by nifedipine or depolarization to +80 mV suppressed the isoproterenol-induced activation of the steady-state BK current. Unlike forskolin, isoproterenol did not change significantly the open probability (NP_o) of single BK channels in the absence of Ca²⁺ sparks and with VDCC inhibited by nifedipine. Isoproterenol elevated Ca²⁺ spark (local intracellular Ca²⁺ release through ryanodine receptors of the sarcoplasmic reticulum) frequency and associated transient BK currents by about 1.4-fold. The data support the concept that in UBSM -adrenoceptor stimulation activates BK channels by elevating Ca²⁺ influx through VDCC and by increasing Ca²⁺ sparks, but not through a Ca²⁺ independent mechanism. This study reveals key regulatory molecular and cellular mechanisms of -adrenergic regulation of BK channels in UBSM that could provide new targets for drugs in the treatment of bladder dysfunction.