Membrane potential (Vm) is tightly controlled in T cells through the regulated flux of ions across the plasma membrane. To investigate the functional role of voltage-dependent (Kv) and calcium-activated (KCa) potassium channels in T cell activation, we compared the effects of two K+ channel blockers, namely kaliotoxin (KTX) and charybdotoxin (CHTX), on Vm, calcium influx, and cell proliferation. KTX potently inhibited Kv (ID50 = 3 nM) but not KCa (ID50 = 5 microM) currents in T cells. Resting T cells exposed to KTX (300 nM) depolarized from -56 mV to -50 mV. KTX had no effect on the transient membrane hyperpolarization that characteristically follows receptor-mediated T cell stimulation. However, T cells stimulated in the presence of KTX subsequently depolarized to -40 mV. KTX also reduced the steady state intracellular free calcium concentration ([Ca2+]i) in stimulated cells by 19% and inhibited T cell proliferation by 35%. CHTX potently inhibited both Kv and KCa currents (ID50 = approximately 1 nM). CHTX (300 nM) depolarized resting T cells to -48 mV, equivalent to the effect observed for KTX. In stimulated T cells, 300 nM CHTX completely blocked the induced hyperpolarization and subsequently depolarized the cells to -21 mV. These effects were associated with a 45% reduction in peak [Ca2+]i, a 60% decrease in steady state [Ca2+]i, and 63% inhibition of T cell proliferation. These results suggest that both Kv and KCa conductances contribute to the underlying mechanisms of T cell activation.