Recent studies have demonstrated that several compounds with diverse structures can activate wild-type cystic fibrosis transmembrane conductance regulator (CFTR) by non-receptor-mediated mechanisms. Some of these compounds have been shown to enhance cAMP-dependent activation of DeltaF508-CFTR. This study was undertaken to compare the mechanisms by which genistein, IBMX, milrinone, 8-cyclopentyl-1, 3-dipropylxanthine (CPX), the benzimidazolone NS004, and calyculin A increase CFTR activity. Our studies demonstrate that, in transfected NIH-3T3 cells, maximal enhancements of forskolin-dependent DeltaF508-CFTR activity are greatest with genistein, IBMX, and NS004. Milrinone, genistein, CPX, NS004, and calyculin A do not increase cellular cAMP. Because forskolin and calyculin A increase in vivo phosphorylation of cAMP binding response element (CREB), the inability of milrinone, genistein, CPX, and NS004 to increase CREB phosphorylation suggests that they do not stimulate protein kinase A or inhibit phosphatase activity. Our data suggest that the mechanisms by which genistein and NS004 activate CFTR differ. We also demonstrate that, in NIH-3T3 cells, IBMX-dependent enhancement of cAMP-dependent CFTR activity is not due to an increase in cellular cAMP and may involve a mechanism like that of genistein.