The role of large conductance Ca2+‐activated K+ (BK) channels in the β3‐adrenoceptor (β3‐AR)‐induced relaxation in rat urinary bladder smooth muscle (UBSM) was studied. BRL 37344, a specific β3‐AR agonist, inhibits spontaneous contractions of isolated UBSM strips. Iberiotoxin, a specific BK channel inhibitor, shifts the BRL 37344 concentration‐response curves for contraction amplitude, muscle force, and tone to the right. Freshly isolated UBSM cells and the perforated patch‐clamp technique were used to determine further the effect of β3‐AR stimulation on the BK channel activity. BRL 37344 increased spontaneous transient outward BK current (STOC) frequency by 46%. In the presence of ryanodine and nifedipine, the single BK channel amplitude and open probability recorded in cell‐attached mode at Vh=0 mV as well as the current‐voltage relationship in response to depolarization steps were not affected by BRL37344. In current‐clamp mode, BRL 37344 caused membrane potential hyperpolarization of about 3 mV. The BRL 37344‐induced hyperpolarization was eliminated by application of iberiotoxin or 1 mM tetraethylammonium. This study provides evidence that in rat UBSM, β3‐ARs and BK channels are functionally coupled so that stimulation of β3‐AR leads to an increase in the BK channel STOC frequency, which causes membrane hyperpolarization and relaxation. Supported by DK070909 to G.V.P.