The neuromodulatory action of chlorothiazide (CTZ) was investigated in isolated rabbit bronchial smooth muscle (BSM) segments contracted with electrical field stimulation (ES). The tissues were placed in organ baths and stimulated with ES frequencies ranging from 1 to 75 Hz. CTZ (10 to 10 M) produced dose-dependent increases in ES-induced contractions. In the presence of 10 M CTZ, the mean ± SEM maximal tension (Tmax) induced by ES increased significantly (p < 0.03) from 292.8 ± 39.5 to 363.0 ± 58.5 g/g tissue. BSM sensitivity to ES, expressed as the log ES frequency producing 50% of Tmax (i.e., log ES50 ) was also increased (p < 0.001) in the presence of CTZ as indicated by a fall in the mean ± SEM log ES50 from 1.05 ± 0.05 to 0.804 ± 0.09 Hz. The potentiating effect of CTZ on ES-induced contractions was independently blocked by either the neurotoxin, tetrodotoxin (4 × 10 M), or the cholinergic antagonist, atropine (10 M). In the presence of CTZ, the mean Tmax response to acetylcholine (ACh) was unaffected, whereas BSM sensitivity to the agonist increased significantly (p < 0.001). On the other hand, the dose-response relationship to carbachol, a cholinergic agonist resistant to cholinesterase degradation, was unaffected by CTZ. In tissues pretreated with 10 M neostigmine, an acetylcholinesterase (AChase) inhibitor, CTZ did not further augment either ES- or ACh-induced contractions. Taken together, these findings suggested that CTZ might be acting as an AChase inhibitor. To further examine the latter hypothesis, we studied the effect of the drug on the enzymatic activity of purified AChase. Between 10 and 10 M CTZ produced dose-dependent decreases in the in vitro activity of purified AChase from a mean ± SEM control activity of 109 ± 5.2 to 45.0 ± 7.5 Rappaport units in the presence of 10 M CTZ (p < 0.01). Our findings provide new evidence that CTZ can augment neurally mediated contraction of isolated rabbit BSM by a mechanism consistent with inhibition of endogenous AChase activity.