Objective: Carbon Monoxide (CO) has been previously described as a regulator of transepithelial Na⁺ transport and epithelial Na⁺ channels (ENaCs). This study questioned whether CO might interfere with Na⁺ self-inhibition - an autoregulatory mechanism of ENaC. Methods: Human a or dENaC together with the b and gsubunits were heterologously expressed in Xenopus laevis oocytes. The activity of the expressed ENaCs was assessed by the two-electrode voltage clamp technique. Results: While the activity of abgENaC was increased by the application of the CO donor molecule CORM-3, dbgENaC was inhibited. These effects were absent in native oocytes, in the presence of the CO-scavenger haemoglobin or when an inactive form of CORM-3 was employed. When zinc – an inhibitor of Na⁺ self-inhibition – was applied to abgENaC, the effects of CORM-3 were abrogated. CORM-3 also diminished the effects of zinc, indicating that both agents share the same target. CORM-3 not only decreased Na⁺ self-inhibition of abgENaC, but also reversed it. dbgENaC, which is known to have a reduced Na⁺ self-inhibition was more sensitive to extracellular Na⁺ after treatment with CORM-3 Conclusion: These data indicate that CO, independently of signalling molecules, interferes with the Na⁺ self-inhibition system of ENaC, leading either to stimulation or inhibition of channel activity, which depends on the subunit composition of ENaC.