We investigated conformational changes occurring in the C-linker and cyclic nucleotide-binding (CNB) domain of CNGA1 channels by analyzing the inhibition induced by thiol-specific reagents in mutant channels Q409C and A414C in the open and closed state. Cd2+ (200 μM) inhibited irreversibly mutant channels Q409C and A414C in the closed but not in the open state. Cd2+ inhibition was abolished in the mutant A414Ccys-free, in the double mutant A414C + C505T and in the tandem construct A414C + C505T/CNGA1, but it was present in the construct A414C + C505cys-free. The cross-linker reagent M-2-M inhibited mutant channel Q409C in the open state. M-2-M inhibition in the open state was abolished in the double mutant Q409C + C505T and in the tandem construct Q409C + C505T/CNGA1. These results show that Cα of C505 in the closed state is located at a distance between 4 and 10.5 Å from the Cα of A414 of the same subunit, but in the open state C505 moves towards Q409 of the same subunit at a distance that ranges from 10.5 to 12.3 Å from Cα of this residue. These results are not consistent with a 3-D structure of the CNGA1 channel homologous to the structure of HCN2 channels either in the open or in the closed state.