The hypothesis that actin interactions account for the signature biophysical properties of cloned epithelial Na+ channels (ENaC) (conductance, ion selectivity, and long mean open and closed times) was tested using planar lipid bilayer reconstitution and patch clamp techniques. We found the following. 1) In bilayers, actin produced a more than 2-fold decrease in single channel conductance, a 5-fold increase in Na+ versus K+ permselectivity, and a substantial increase in mean open and closed times of wild-type αβγ-rENaC but had no effect on a mutant form of rENaC in which the majority of the C terminus of the α subunit was deleted (α(R613X)βγ- rENaC). 2) When α(R613X)βγ-rENaC was heterologously expressed in oocytes and single channels examined by patch clamp, 12.5-pS channels of relatively low cation permeability were recorded. These characteristics were identical to those recorded in bilayers for either α(R613X)βγ-rENaC or wild-type αβγ-rENaC in the absence of actin. Moreover, we show that rENaC subunits tightly associate, forming either homo- or heteromeric complexes when prepared by in vitro translation or when expressed in oocytes. Finally, we show that α-rENaC is properly assembled but retained in the endoplasmic reticulum compartment. We conclude that actin subserves an important regulatory function for ENaC and that planar bilayers are an appropriate system in which to study the biophysical and regulatory properties of these cloned channels.