Maintenance by the endothelium of a semi-permeable barrier is critically important in the exchange of oxygen and carbon dioxide in the lung. Intracellular free Ca2+ ([Ca2+]i) and cAMP are principal determinants of endothelial cell barrier function through their mutually opposing actions on endothelial retraction. However, details of the mechanisms of this antagonism are lacking. The recent discovery that certain adenylyl cyclases (EC 4.6.1.1) could be acutely inhibited by Ca2+ in the intracellular concentration range provided one possible mechanism whereby elevated [Ca2+]i could decrease cAMP content. This possibility was explored in pulmonary artery endothelial cells. The results indicate that a type VI Ca(2+)-inhibitable adenylyl cyclase exists in pulmonary artery endothelial cells and is modulated by physiological changes in [Ca2+]i. Furthermore, the results suggest the inverse relationship between [Ca2+]i and cAMP that is established by Ca(2+)-inhibitable adenylyl cyclase plays a critical role in modulating pulmonary artery endothelial cell permeability. These data provide evidence that susceptibility to inhibition of adenylyl cyclase by Ca2+ can be exploited in modulating a central physiological process.