1. Cytochalasin D (CD; 5 microM) readily stimulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel activity in cell-attached and whole-cell patch recordings from 3T3 fibroblasts expressing recombinant CFTR but not in mock-transfected cells. CD-stimulated currents were indistinguishable from those evoked by forskolin stimulation. Kinetic analysis of CFTR gating showed identical channel behaviour independent of the agonist used. 2. To elucidate the mechanism of action of CD we tested its effects on cAMP, protein kinase A, and the CFTR itself during CD stimulation. In contrast to forskolin treatment, CD did not increase cellular cAMP content. 3. A direct interaction of CD with the CFTR was ruled out because CD showed no effect on CFTR in excised inside-out patches. 4. CD effects were fully blocked when the cellular protein kinase A was inhibited by treatment of cells with RpcAMPS in cell-attached patches or when protein kinase inhibitor peptide was dialysed into cells in whole-cell experiments. 5. Addition of G-actin to excised patches had no effects on CFTR. 6. We conclude that the stimulatory effect of CD is cAMP independent, but needs a functional protein kinase A in order to activate the CFTR. We propose that cytochalasin D activates CFTR by releasing a cellular inhibitor, e.g. a phosphatase, that is held in place by F-actin.