Chloride channel activity of cystic fibrosis transmembrane conductance regulator (CFTR) requires activation of protein kinase A (PKA) by 3'-5'-cyclic adenosine monophosphate (cAMP). The level of cAMP is controlled by the balance between cAMP synthesis and hydrolysis by adenylate cyclase and phosphodiesterases (PDEs), respectively. CFTR channel activity appears to be most sensitive to the activity of type III cyclic nucleotide PDEs in Calu-3 and 16HBE cells, both derived from airway epithelium and expressing wild-type CFTR. Type III PDEs can be identified by their sensitivity to specific inhibitors such as milrinone and amrinone. In Calu-3 cells, specific inhibition of type III PDEs increased chloride efflux up to 13.7-fold, whereas neither rolipram nor Ro20-1724 (type IV PDE inhibitors) nor 3-isobutyl-1-methylxanthine (IBMX, a nonspecific PDE inhibitor) elicited significant increases. None of these compounds had an appreciable effect on total cellular cAMP levels, yet the effects of milrinone and amrinone on chloride efflux were blocked by treatment of cells with Rp-cAMPS, a cAMP analog that inhibits PKA at the site of cAMP binding. Similarly, H-8, an inhibitor of PKA, reduced milrinone-stimulated chloride efflux, indicating that efflux is mediated through the cAMP/PKA pathway. Whole-cell patch clamp analysis revealed that milrinone generated chloride conductances with properties consistent with those of CFTR. Milrinone elicited chloride currents in a dose-dependent manner and induced CFTR activity in the absence of adenylate cyclase agonists. These data suggest that type III PDEs are specifically involved in CFTR activation in airway epithelial cells and that PDE regulation of CFTR may involve subcellular compartments of cAMP.