According to the two state receptor model, the β2‐adrenergic receptor (β2‐AR) isomerizes between an inactive state and a constitutively active state, which couples to the stimulatory G‐protein in the absence of agonist. In bovine tracheal smooth muscle (BTSM), we investigated the effect of short and long term β2‐AR activation by fenoterol on constitutive receptor activity. Preincubation of the BTSM strips for 5 min, 30 min and 18 h with 10 μM fenoterol, followed by extensive washout (3 h, 37°C), caused a rapid and time‐dependent inhibition of KCl‐induced contraction, reaching 68±10, 51±6 and 46±4% of control, respectively, at 40 mM KCl (P<0.05 all). At all time points, the EC50 values to KCl were significantly reduced as well. Preincubation of BTSM with 0.1, 1.0 and 10 μM fenoterol during 18 h caused a concentration‐dependent decrease of the 40 mM KCl response to 70±5, 47±12 and 43±9% of control, respectively (P<0.05 all). The reduced KCl contractions were reversed in the presence of 1 μM timolol. Moreover, the sensitivity to KCl in the presence of timolol was enhanced after fenoterol incubation. Inverse agonism was also found for other β‐blockers, with a rank order of efficacy of pindolol timolol=propranolol>alprenololsotalol>labetalol. At 25 mM KCl‐induced tone, the contraction induced by cumulative timolol administration was competitively antagonized by the less efficacious inverse agonist labetalol, indicating that the fenoterol‐induced effects cannot be explained by residual β‐agonist binding. In conclusion, fenoterol treatment of BTSM causes a time‐ and concentration‐dependent development of constitutive β2‐AR activity, which can be reversed by various inverse agonists. The β‐agonist‐induced changes could represent a novel regulation mechanism of β2‐AR activity. British Journal of Pharmacology (2000) 131, 915–920; doi:10.1038/sj.bjp.0703664