We have studied activation by phorbol derivatives of TRPV4 channels, the human VRL-2, and murine TRP12 channels, which are highly homologous to the human VR-OAC, and the human and murine OTRPC4 channel. 4α-Phorbol 12,13-didecanoate (4α-PDD) induced an increase in intracellular Ca2+ concentration, [Ca2+]i, in 1321N1 cells stably transfected with human VRL-2 (hVRL-2.1321N1) or HEK-293 cells transiently transfected with murine TRP12, but not in nontransfected or mock-transfected cells. Concomitantly with the increase in [Ca2+]i, 4α-PDD activated an outwardly rectifying cation channel with an Eisenman IV permeation sequence for monovalent cations that is Ca2+-permeable withP Ca/P Na = 5.8. Phorbol 12-myristate 13-acetate also induced an increase in [Ca2+]i but was ∼50 times less effective than 4α-PDD. EC50 for Ca2+ increase and current activation was nearly identical (pEC50 ∼ 6.7). Similar effects were observed in freshly isolated mouse aorta endothelial cells which express TRP12 endogenously. By using 4α-PDD as a tool to stimulate TRP12, we showed that activation of this channel is modulated by [Ca2+]i; an increase in [Ca2+]i inhibits the channel with an IC50 of 406 nm. Ruthenium Red at a concentration of 1 μm completely blocks inward currents at −80 mV but has a smaller effect on outward currents likely indicating a voltage dependent channel block. We concluded that the phorbol derivatives activate TRPV4 (VR-OAC, VRL-2, OTRPC4, TRP12) independently from protein kinase C, in a manner consistent with direct agonist gating of the channel.