The amiloride–sensitive Na⁺ channel, found in the apical membrane of polarized epithelia form the rate–limiting step for Na⁺ reabsorption and fluid homeostasis [1]. We have produced a stably transfected clone of the H441 cell line that contain low levels of enhanced green fluorescent protein (EGFP)–labelled human αENaC subunit. We have confirmed that the appropriate EGFP–αENaC RNA and protein are expressed in the generated cell line. We have also determined the subcellular localization using confocal microscopy and measured functional amiloride-sensitive Na⁺ transport in the presence and absence of forskolin. H441 cell monolayers were cultured on permeable supports for 7 days at air interface, mounted in Ussing chambers and spontaneous short circuit current (I_sc) was measured by voltage clamping the monolayers at zero. Statistical analysis was carried out using Student’s t-test where p values of < 0.05 were considered significant. Data are presented as mean ± SEM. Cells expressing EGFP–αENaC (αC3) exhibited an I_sc of 16.3 ± 0.7 μA cm^-2 compared to 6.4 ± 0.7 μA cm^-2 in cells expressing EGFP alone (p<0.001, n=3). Forskolin stimulated an increase in I_sc in clone αC3 of 7.0 ± 2.1 μA cm^-2 which was significantly greater than cells expressing EGFP alone (0.09 ±0.08 μA.cm^-2, p<0.05, n=3). Application of 10μM amiloride to the monolayers demonstrated that amiloride–sensitive I_sc was also significantly higher in αC3 cells than in those transfected with EGFP alone (10.7 ± 0.84 and 3.0 ± 0.4 μA.cm^-2 respectively p=0.001, n=3). Composite images from 10 μm sections of forskolin treated cells showed increased EGFP fluorescence in the upper (apical) compartment of the αC3 clone which was not evident in cells expressing EGFP alone. Taken together, these data show that stable expression of human EGFP–αENaC subunits in H441 cells results in functional amiloride–sensitive Na⁺ channels. Treatment with forskolin increased function and was associated with increased fluorescence in the upper compartment of the cell.