Organophosphates induce bronchoobstruction in guinea pigs, and salbutamol only transiently reverses this effect, suggesting that it triggers additional obstructive mechanisms. To further explore this phenomenon, in vivo and in vitro experiments were performed. In in vivo experiments, parathion caused a progressive bronchoobstruction until a plateau was reached. Administration of salbutamol during this plateau decreased bronchoobstruction up to 22% in the first 5 min, but thereafter airway obstruction rose again. Aminophylline caused a sustained decrement (71%) of the parathion-induced bronchoobstruction. In in vitro studies, paraoxon produced a sustained contraction of tracheal rings, which was fully blocked by atropine but not by TTX, -conotoxin (CTX) or epithelium removal. During the paraoxon-induced contraction, salbutamol caused a temporary relaxation of ~50% followed by a partial re-contraction. This paradoxical re-contraction was avoided by the M₂- or NK₁-receptor antagonists (methoctramine or AF-DX 116, and L-732,138, respectively), accompanied by a long lasting relaxation. Substance P, and at lesser extent ACh, released from tracheal rings during 60-min incubation with paraoxon or physostigmine, respectively, were significantly increased when salbutamol was administered in the second half of this period. In myocytes, paraoxon did not produce any change in the intracellular Ca²⁺ basal levels. Our results suggested that: 1) organophosphates caused smooth muscle contraction by accumulation of ACh released through a TTX- and CTX-resistant mechanism, 2) during such contraction, salbutamol relaxation is functionally antagonized by the stimulation of M₂ receptors, and 3) after this transient salbutamol-induced relaxation, a paradoxical contraction ensues due to the subsequent release of substance P.