We present evidence that activity of native Kv1.3 channels in human T lymphocytes can be increased by inhibiting phosphatases [using okadaic acid (OA)] or by activating protein kinase A (PKA). OA increased the maximal conductance (Gmax) by 40% and shifted the voltage dependence of activation and inactivation, resulting in a significant increase in window current around the normal membrane potential. PKA inhibition [using the PKA inhibitor peptide PKI-(5-24)] decreased Gmax by 43%, whereas PKA activation [by the Sp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Sp-cAMPS)] increased Gmax by 60% and shifted the inactivation curve, producing an increase in the window current. These results are consistent with our previously published work using cell-attached patches but differ from some studies of Kv1.3. Because we previously reported a similar upregulation by protein kinase C (PKC) activation in these cells, we tested whether the PKA and PKC effects were additive. Our results suggest that PKC-dependent phosphorylation acts as a master switch, inasmuch as calphostin C greatly inhibited the current even after Sp-cAMPS, OA, or PKC activation was used to increase protein phosphorylation. Inasmuch as phosphorylation by both kinases (phorbol ester followed by Sp-cAMPS) abrogated the effects of either kinase alone, our results support the view that Kv1.3 is regulated in a complex manner by serine/threonine phosphorylation.