The once a day muscarinic antagonist tiotropium bromide (SpirivaTM) is an established bronchodilator therapy for the treatment of COPD. There have been few reports documenting antagonist affinity in functional assays in the human isolated bronchus. In this report we have studied the effect of incubation time on the functional antagonist potency of tiotropium compared with ipratropium and atropine. Bronchial rings of internal diameter 1-7mm were suspended in 15ml tissue baths under an initial resting tension of 2g and bathed in warmed (37°C), aerated (95%O2/5%CO2) Krebs solution. Following a 2h equilibration period two cumulative concentration effect curves (CEC) to carbachol (CCh) were constructed 1h apart. The second CEC served as the control curve. Antagonists were incubated with the tissues for 1h, 2h or 4h before a 3rd CEC to CCh was carried out. Concentration ratios (CR) were calculated and used to calculate pKB values (Gaddum 1957). In some experiments only 2 concentrations of the surmountable antagonists could be tested therefore the average pKB value was quoted. For non-surmountable antagonists a single pKB value was calculated at the lowest effective (CR>2.0) concentration. Data for pKB values are arithmetic means ±95% confidence intervals (C.I.). Statistical analysis was performed using unpaired t test. Atropine and ipratropium bromide produced concentration related rightward shifts in CCh CECs with no suppression of agonist maximum responses at the times and concentrations studied. Atropine gave an average pKB of 9.1(8.7-9.5) n=4 and 9.0 (8.5-9.4) n=5 following 1h and 2h incubation respectively. These values were not significantly different (p>0.05). Ipratropium gave average pKB values of 9.7(9.0-10.4)n=3 at 1h and 9.0(8.4-9.6)n=3 at 4h (p0.05). Atropine and ipratropium were surmountable antagonists at the concentrations and incubation times studied. Potency was not increased on extended incubation. Tiotropium was a non-surmountable antagonist and its pKB was not increased by extending the incubation time from 1h to 4h. Tiotropium was more potent than atropine at 1h and 2h and ipratropium at 4h (p<0.05). Incubation times of 1h were sufficient for the system to be at equilibrium for tiotropium.