The relaxant effects to the β‐adrenoceptor agonists isoprenaline, adrenaline, noradrenaline, RO363, procaterol and fenoterol were investigated in carbachol‐contracted mouse isolated tracheal preparations. The order of potencies for those β‐adrenoceptor agonists that induced full relaxation of carbachol‐contracted mouse tracheal preparations was isoprenaline > RO363 > noradrenaline = adrenaline > fenoterol. The EC50 value of isoprenaline for relaxation was 46 nm. The β1‐adrenoceptor‐selective agonist, RO363, was ten times more potent than the β2‐adrenoceptor‐selective agonist, fenoterol. The highly β2‐adrenoceptor‐selective agonist procaterol was a partial relaxant and induced only 28 ± 4% relaxation. Relaxations induced by noradrenaline and isoprenaline were not significantly affected by the neuronal uptake inhibitor, cocaine (10 μm) or by the extraneuronal uptake inhibitor, deoxycorticosterone acetate (25 μm) respectively. The α‐adrenoceptor agonist methoxamine induced no observable elevation of mouse tracheal smooth muscle tone. Schild plots for the β‐adrenoceptor antagonists, atenolol and betaxolol (β1‐adrenoceptor‐selective) and ICI 118,551 (β2‐adrenoceptor‐selective) were linear, with slope values approaching unity. Mean pA2 values derived for atenolol, betaxolol and ICI 118,551 for antagonism of β‐adrenoceptor‐mediated relaxation were 7.1, 8.4 and 7.2, respectively. These data were independent of the use of isoprenaline or noradrenaline as the agonist. These findings indicate that β‐adrenoceptor‐mediated relaxations of mouse isolated trachea occur predominantly through activation of β1‐adrenoceptors.