Cyclic nucleotide-gated (CNG) channels and K+ channels have a significant sequence identity and are thought to share a similar 3D structure. K+ channels can accommodate simultaneously two or three permeating ions inside their pore and therefore are referred to as multi-ion channels. Also CNGA1 channels are multi-ion channels, as they exhibit an anomalous mole fraction effect (AMFE) in the presence of mixtures of 110 mM Li+ and Cs+ on the cytoplasmic side of the membrane. Several observations have identified the ring of Glu363 in the outer vestibule of the pore as one of the binding sites within the pore of CNGA1 channels. In the present work we identify a second binding site in the selectivity filter of CNGA1 channels controlling AMFE. Here, we show also that Cs+ ions at the intracellular side of the membrane block the entry of Na+ ions. This blockage is almost completely removed at high hyperpolarized voltages as expected if the Cs+ blocking site is located within the transmembrane electric field. Indeed, mutagenesis experiments show that the block is relieved when Thr359 and Thr360 at the intracellular entrance of the selectivity filter are replaced with an alanine. In T359A mutant channels AMFE in the presence of intracellular mixtures of Li+ and Cs+ is still present but is abolished in T360A mutant channels. These results suggest that the ring of Thr360 at the intracellular entrance of the selectivity filter forms another ion binding site in the CNGA1 channel. The two binding sites composed of the rings of Glu363 and Thr360 are not independent; in fact they mediate a powerful coupling between permeation and gating, a specific aspect of CNG channels.