Ion channels directly activated by the binding of cGMP mediate the electrical response to light in rod photoreceptors. Here, we identify a region of the channel associated with the activation gate using potentiation by intracellular Ni2+. Low concentrations of Ni2+ caused a dramatic increase in the response of rod channels expressed in Xenopus oocytes to both cGMP and cAMP. Whereas saturating cAMP normally activated less than 1% of the channels, Ni2+ increased the cAMP potency nearly 50-fold. Ni2+ did not produce potentiation in the related channel from the olfactory epithelium. We localized the Ni(2+)-binding site to a histidine residue in the putative intracellular mouth of the rod channel (H420). We propose a mechanism for potentiation in which Ni2+ binds to H420 primarily when the channel is open, stabilizing the open conformation. These experiments suggest that H420 is associated with the activation gate.