Taking a big breath is known to reverse bronchoconstriction induced by bronchochallenge in healthy subjects; this bronchodilatory effect of deep inspiration (DI) is diminished in asthmatics. The mechanism underlying the DI effect is not clear. Observations from experiments using isolated airway smooth muscle (ASM) preparations and airway segments suggest that straining of ASM due to DI could lead to bronchodilation, possibly due to strain-induced reduction in ASM contractility. However, factors external to the lung cannot be excluded as potential causes for the DI effect. Neural reflex initiated by stretch receptors in the lung are known to inhibit the broncho-motor tone and enhance vasodilatation; the former directly reduces airway resistance, and the latter facilitates removal of contractile agonists through the bronchial circulation. If the DI effect is solely mediated by factors extrinsic to the lung, the DI effect would be absent in isolated, non-perfused lungs. Here we examined the DI effect in freshly isolated, non-perfused sheep lungs. We found that imposition of DI on isolated lungs resulted in significant bronchodilation, and this DI effect was present only after the lungs were challenged with a contractile agonist (acetylcholine or histamine), and the effect was independent of the difference in lung volume observed pre- and post-DI. We conclude that a significant portion of the bronchodilatory DI-effect stems from factors internal to the lung related to the activation of ASM.