In this study, we applied high-performance liquid chromatography with electrochemical detection (HPLC-EC) to the measurement of acetylcholine (ACh) release from nerve endings in guinea pig tracheal smooth muscle. We also tested for muscarinic inhibitory regulation of ACh release in this species, which is widely used for studies of airway neural control. Clip-connected segments of the posterior membrane of the guinea pig trachea were mounted in organ baths between stimulating electrodes and incubated in Krebs-Henseleit buffer containing (in microM) 10 indomethacin, 1 neostigmine, 1 phentolamine, and 1 propranolol. To measure ACh, the bath was emptied and aliquots of buffer were injected directly into the HPLC-EC; the lower limit of detection was 1 pmol/200 microliters sample. Electrical field stimulation (EFS) at 5 Hz for 10 or 30 min increased ACh release from 1.8 +/- 1.4 (SE) to 6.2 +/- 1.3 pmol.mg protein-1.min-1 (n = 15). The effect of atropine was examined by comparing amounts of ACh released by EFS before and after exposure to either atropine (0.3 microM) or vehicle. Before atropine treatment, EFS-evoked ACh release was 4.9 +/- 0.6 pmol.mg protein-1.min-1; after atropine exposure, EFS-evoked release of ACh increased significantly to 15.0 +/- 2.2 pmol.mg protein-1.min-1 (n = 11; P less than 0.05). Corresponding values before and during exposure to vehicle were 9.3 +/- 4.4 and 10.7 +/- 4.7 pmol.mg protein-1.min-1, respectively. The ratios of the changes in EFS-evoked ACh release were 3.1 +/- 0.3 and 1.3 +/- 0.1 in atropine-treated and vehicle-treated groups, respectively (P less than 0.05). We conclude that HPLC-EC is a reliable and sensitive technique for the detection of EFS-evoked release of ACh from clip-connected segments of guinea pig tracheal smooth muscle.