Dequalinium has recently been reported to block CNGA1 and CNGA2 channels expressed in Xenopus laevis. Using the inside-out configuration of the patch-clamp technique, we examined the effects of dequalinium on rat olfactory CNGA2 channels expressed in human embryonic kidney (HEK293) cells and studied aspects of its molecular mechanism of action. We found that cytoplasmic dequalinium blocked wild-type (WT) CNGA2 channels in a voltage-dependent manner with an IC(50) of approximately 1.3 muM at a V(m) of + 60 mV, and an effective fractional charge, zdelta, of +0.8 (z=2, delta=+0.4), suggesting that cytoplasmic dequalinium interacts with a binding site that is about two fifths of the way along the membrane electric field (from the intracellular side). Neutralizing the negatively charged pore lining glutamate acid residue (E342Q) still allows effective channel block by cytoplasmic dequalinium with an IC(50) of approximately 2.2 muM at a V(m) of +60 mV but now having a zdelta of +0.1 (delta=+0.05), indicating a profoundly decreased level of voltage-dependence. In addition, by comparing the extent of block under different levels of channel activation, we show that the block by cytoplasmic dequalinium displayed clear state-dependence in WT channels by interacting predominantly with the closed channel, whereas the block in E342Q channels was state-independent. Application of dequalinium to the external membrane surface also blocked currents through WT channels and the E342Q mutation significantly increased the IC(50) for external block approximately fivefold. These results confirm dequalinium as a potent, voltage-dependent and state-dependent blocker of cyclic-nucleotide-gated channels, and show that neutralization of the E342 residue profoundly affects the block by both cytoplasmic and external application of dequalinium.