Pleural effusions are commonly clinical disorders, resulting from the imbalance between pleural fluid turnover and re-absorption. The mechanisms underlying pleural fluid clearance across the mesothelium remain to be elucidated. We hypothesized that ENaC is expressed and forms the molecular basis of the amiloride-sensitive resistance in human mesothelial cells. Our RT-PCR results showed that four ENaC subunits, namely, [alpha], [beta], [gamma], and two [delta] ENaC subunits are expressed in human primary pleural mesothelial cells, a human mesothelioma cell line (M9K), and mouse pleural tissue. In addition, Western blotting and immunofluorescence microscopy studies revealed that [alpha], [beta], [gamma], and [delta] ENaC subunits are expressed in primary human mesothelial cells and M9K cells at the protein level. An amiloride-inhibitable short-circuit current was detected in M9K monolayers and mouse pleural tissues when mounted in Ussing chambers. Whole-cell patch clamp recordings showed an ENaC-like channel with an amiloride IC₅₀ of 12 M in M9K cells. This cation channel has a high affinity for extracellular Na+ ions (Km: 53mM). The ion selectivity of this channel to cations follows the same order as ENaC: Li⁺ > Na⁺ > K⁺. The unitary Li⁺ conductance was 15 pS in on-cell patches. Four ENaC subunits form a functional Na⁺ channels when co-injected into Xenopus oocytes. Furthermore, we found that both forskolin and cGMP increased the short-circuit currents in mouse pleural tissues. Taken together, our data demonstrate that the ENaC channels are biochemically and functionally expressed in human pleural mesothelial cells, and can be up-regulated by cAMP and cGMP.