A variety of mechanisms have been proposed for the regulation of ion channel molecules. As integral membrane proteins, ion channels may interact with the cytoskeleton. Regulation of channels by the actin network may therefore be important. In the present study we used cytochalasin D and exogenous actin to test this possibility. The Cl- channel of the apical membrane of renal proximal epithelium was detected in its active state after prolonged depolarization. Within 6 sec after its addition, cytochalasin D (0.05 microgram/ml) significantly decreased the number of open channels and mean open probability (NPo) of the Cl- channel. Colchicine (1 mM), which affects microtubules, did not influence channel activation. Cytochalasin D is known to not only disrupt the F-actin network but to inhibit polymerization of F-actin as well. The latter effect is also produced by DNaseI. Cytochalasin D, but not DNaseI, inactivated Cl- channels in cell-free membrane patches, suggesting that cytochalasin D inactivated the channel by disrupting the actin network. Cytochalasin D appeared to specifically affect the channel, as opposed to membrane permeability, since only the activated whole-cell Cl- currents were altered by cytochalasin D. Addition of actin polymer, but not actin monomer, reactivated the cytochalasin-D-depressed channel. Thus, repair of the disrupted F-actin network with actin polymer apparently restored the activity and number of open Cl- channels. We therefore conclude that the F-actin network interacts with and possibly regulates the Cl- channel of renal proximal tubule epithelia.