Plasma membrane potassium (K⁺) channels are required for cell proliferation. Evidence is growing that K⁺ channels play a central role in the development and growth of human cancer. Here we examine the contribution and the mechanism by which K⁺ channels control proliferation of T₈₄ human colonic carcinoma cells. Numerous K⁺ channels are expressed in T₈₄ cells, but only voltage-gated K⁺ (Kv) channels influenced proliferation. A number of Kv channel inhibitors reduced DNA synthesis and cell number, without exerting apoptotic or toxic effects. Expression of several Kv channels, such as EagI, Kv 3.4 and Kv 1.5, was detected in patch clamp experiments and in fluorescence-based assays using a voltage sensitive dye. The contribution of EagI channels to proliferation was confirmed by siRNA, which abolished EagI activity and inhibited cell growth. Inhibition of Kv channels did not interfere with the ability of T₈₄ cells to regulate their cell vol, but it restricted intracellular pH regulation. In addition, inhibitors of Kv channels, as well as siRNA for EagI, attenuated intracellular Ca²⁺ signaling. The data suggest that Kv channels control proliferation of colonic cancer cells by affecting intracellular pH and Ca²⁺ signaling.--Spitzner, M., Ousingsawat, J., Scheidt, K., Kunzelmann, K., Schreiber, R. Voltage gated K⁺ channels support proliferation of colonic carcinoma cells.