Anomalous mole–fraction effects (AMFE) were studied, using the inside–out configuration of the patchclamp technique, in both recombinant wild–type α–homomeric rat olfactory adenosine 3′,5′–cyclic monophosphate (cAMP)–gated channels (rOCNC1) expressed in human embryonic kidney cells (HEK 293) and native cyclic nucleotide–gated (CNG) channels in acutely isolated rat olfactory receptor neurons. Single–channel and macroscopic currents were activated by 200 μM and 500 μM cAMP, respectively. Macroscopic currents, measured with mixtures of Na+–NH4+ or Cs+–Li+ in the cytoplasmic bathing solution, displayed AMFE in the rOCNC1 channels at both positive and negative membrane potentials. The rOCNC1 single–channel conductance showed a distinct minimum (or maximum) in an 80% Na+–20% NH4+ mixture (or a 60% Cs+–40% Li+ mixture), but only at positive membrane potentials. Macroscopic measurements in native olfactory CNG channels with mixtures of Na+–NH4+ indicated similar AMFE. These results suggest that both native CNG channels and recombinant α–homomeric channels allow several ions to be present simultaneously within the channel pore. They also further validate the dominant role of the α–subunit in permeation through these channels, provide the first evidence to suggest that rOCNC1 channels have multi–ion properties and further justify the use of the rOCNC1 channel as an effective model for structure–function studies of ion permeation and selectivity in olfactory CNG channels.