In response to depolarization of the membrane potential, Shaker K+ channels undergo a series of voltage-dependent conformational changes, from resting to open conformations followed by a rapid transition into a long-lived closed conformation, the N-type inactivated state. Application of phosphatases to the cytoplasmic side of Shaker channels in excised inside-out patches slows N-type inactivation gating. Subsequent application of the purified catalytic subunit of the cAMP-dependent protein kinase (PKA) and ATP reverses the effect, accelerating N-type inactivation back to its initial rapid rate. Macroscopic and single-channel experiments indicate that N-type inactivation is selectively modulated. There was little or no effect on the voltage dependence and kinetics of activation. Comparison of site-directed mutant channels shows that a C-terminal consensus site for PKA phosphorylation is responsible for the modulation. Since a cell's integrative characteristics can be determined by the rate of inactivation of its voltage-dependent channels, modulation of these rates by phosphorylation is likely to have functional consequences.