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The molecular events underlying CFTR-ENaC coupling, two major transport proteins implicated in pathophysiology of Cystic Fibrosis, is obscure. In an attempt to examine the intermolecular interaction of these proteins we used fluorescence resonance energy transfer (FRET) microscopy. FRET is distance-dependent imaging approach to measure protein-protein interactions. To measure FRET we used acceptor bleaching, and monitored the donor's fluorescence lifetime by frequency domain fluorescence lifetime imaging microscopy (FLIM). These FRET measurements were complemented by coimmunoprecipitation experiments. Apparent FRET efficiencies (E) averaged ∼8% but values as high as ∼17% were observed, significant when compared to the negative control and comparable to E reported in other FRET studies. No appreciable FRET signal was observed when ECFP-CICI was co-transfected with αβγ-ENaC (<1%). When CFTR and ENaC subunits were over-expressed in HEK293T cells, we found that β-ENaC could be co-immunoprecipitated with CFTR. Under the same condition, we were unable to co-immunoprecipitate β-ENaC with ECFP-CIC1. Our results place CFTR and ENaC in close proximity to each other, suggestive of direct interaction between these two proteins. [ABSTRACT FROM AUTHOR]