Respiratory pathology research is limited by the number of appropriate multicellular models suitable for studying mechanical properties and signaling pathways that are involved in airway responsiveness. In this study, the electrophysiological and pharmacomechanical properties of organ-cultured explants derived from normal guinea pig bronchi and trachea were investigated. The explants maintained their basic histological phenotype but became hyperreactive to excitatory (muscarinic, histaminergic, serotinergic, and thromboxane receptor agonists, 60 mM KCl) and inhibitory (norepinephrine, isoproterenol) stimuli within the first 3 days in culture, with or without serum in the culture medium. Indomethacin pretreatment did not modify the spasmogen responses of the explant. The onset of this intrinsic overreactivity was highly dependent on the initial presence of epithelium, took 3 days to reach its maximum, and lasted over several days (days 3 to 7). Removal of Ca2+ from the bathing solution initially normalized the inotropic responses of the cultured versus freshly isolated airway tissues. However, the responses to repetitive carbachol challenges in the absence of Ca2+ displayed a slower inactivation in the cultured explants compared to fresh tissues. Smooth muscle resting membrane potential and potassium-induced depolarizations were unaffected by organ culture. Immunohistochemical analyses revealed the presence of apoptotic bodies in the submucosa and epithelial layers, but none in the smooth muscle layer of cultured airways. These functional and histological findings may prove useful in understanding signaling processes involved in tissue hyperresponsiveness related to asthma.