1. Using whole cell voltage-clamp recordings, the guanylyl cyclase inhibitor LY83583 [6-(phenylamino)-5,8-quinolinedione] is shown to act as a potent blocker of cyclic nucleotide-gated (CNG) channels in isolated olfactory receptor neurons (ORNs) of the tiger salamander. 2. Under our experimental conditions, onset of the blockade by LY83583 occurs on the time scale of seconds and is completely reversed upon wash-out of the drug. Dose-response curves reveal a Kd of 1.4 microM (at -60 mV). Other data suggest that LY83583 acts within the CNG channel pore and that the channels must be in an activated state before the drug can exert its effect. 3. It appears that LY83583 can act on both CNG channels and soluble guanylyl cyclase (sGC) and that these two effects can be distinguished by their different recovery behaviors. The LY83583-induced blockade of CNG channels activated directly by guanosine 3',5' cyclic monophosphate (cGMP) is rapidly reversible (with a recovery time constant of approximately 3 s), whereas previous results have shown that no recovery is obtained during minute-long washing periods when the channels are activated indirectly through exogenous carbon monoxide application, which acts as a stimulator of sGC in ORNs. 4. LY83583 appears to be a novel and useful agent in examining neural functions due to CNG channel responses.