We have examined the modulation by internal protons of cyclic nucleotide–gated (CNG) channels cloned from bovine olfactory receptor cells and retinal rods. CNG channels were studied in excised inside–out membrane patches from Xenopus laevis oocytes previously injected with the mRNA encoding for the subunit~1 of olfactory or rod channels. Channels were activated by cGMP or cAMP, and currents as a function of cyclic nucleotide concentrations were measured as pHi varied between 7.6 and 5.0. Increasing internal proton concentrations caused a partial blockage of the single–channel current, consistent with protonation of a single acidic site with a pK1 of 4.5–4.7, both in rod and in olfactory CNG channels. Channel gating properties were also affected by internal protons. The open probability at low cyclic nucleotide concentrations was greatly increased by lowering pHi, and the increase was larger when channels were activated by cAMP than by cGMP. Therefore, internal protons affected both channel permeation and gating properties, causing a reduction in single–channel current and an increase in open probability. These effects are likely to be caused by different titratable groups on the channel.