The direct detrusor relaxant effect of β3-adrenoceptor agonists as a primary mechanism to improve overactive bladder symptoms has been questioned. Among other targets, activation of β3-adrenoceptors down-modulate nerve-evoked acetylcholine (ACh) release, but there is insufficient evidence for the presence of these receptors on bladder cholinergic nerve terminals. Our hypothesis is that adenosine formed from the catabolism of cyclic AMP in the detrusor may act as a retrograde messenger via prejunctional A1 receptors to explain inhibition of cholinergic activity by β3-adrenoceptors. Isoprenaline (1 µM) decreased [3H]ACh release from stimulated (10 Hz, 200 pulses) human (-47±5%) and rat (-38±1%) detrusor strips. Mirabegron (0.1 µM, -53±8%) and CL316,243 (1 µM, -37±7%) mimicked isoprenaline (1 µM) inhibition and their effects were prevented by blocking β3-adrenoceptors with L748,337 (30 nM) and SR59230A (100 nM), respectively in human and rat detrusor. Mirabegron and isoprenaline increased extracellular adenosine in the detrusor. Blockage of A1 receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 100 nM) or the equilibrative nucleoside transporters (ENT) with dipyridamole (0.5 µM) prevented mirabegron and isoprenaline inhibitory effects. Dipyridamole prevented isoprenaline-induced adenosine outflow from the rat detrusor and this effect was mimicked by the ENT1 inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI, 30 µM). Cystometry recordings in anaesthetized rats demonstrated that SR59230A, DPCPX, dipyridamole and NBTI reversed the decrease in the voiding frequency caused by isoprenaline (0.1-1000 nM). Data suggest that inhibition of cholinergic neurotransmission by β3-adrenoceptors results from adenosine release via equilibrative nucleoside transporters and prejunctional A1 receptors stimulation in human and rat urinary bladder.