Kv1.1/Kvbeta1.1 (alphabeta) K+ channel expressed in Xenopus oocytes was shown to have a fast inactivating current component. The fraction of this component (extent of inactivation) is increased by microfilament disruption induced by cytochalasins or by phosphorylation of the alpha subunit at Ser-446, which impairs the interaction of the channel with microfilaments. The relevant sites of interaction on the channel molecules have not been identified. Using a phosphorylation-deficient mutant of alpha, S446A, to ensure maximal basal interaction of the channel with the cytoskeleton, we show that one relevant site is the end of the C terminus of alpha. Truncation of the last six amino acids resulted in alphabeta channels with an extent of inactivation up to 2.5-fold larger and its further enhancement by cytochalasins being reduced 2-fold. The wild-type channels exhibited strong inactivation, which could not be markedly increased either by cytochalasins or by the C-terminal mutations, indicating that the interaction of the wild-type channels with microfilaments was minimal to begin with, presumably because of extensive basal phosphorylation. Since the C-terminal end of Kv1.1 was shown to participate in channel clustering via an interaction with members of the PSD-95 family of proteins, we propose that a similar interaction with an endogenous protein takes place, contributing to channel connection to the oocyte cytoskeleton. This is the first report to assign a modulatory role to such an interaction: together with the state of phosphorylation of the channel, it regulates the extent of inactivation conferred by the beta subunit.