Objective: The precise subunit composition of functional protein complexes is often unknown. For example, although the epithelial sodium channel (ENaC) was first described as a two aENaC, one bENaC and one g ENaC subunit heterotetrameric complex, higher order complexes have been proposed as well. The highly related protein complex ASIC1 was recently shown to assemble as a trimer. Our study aims to improve the methodology for determining subunit composition of functional multiprotein complexes. Methods: A sequential bioluminescence resonance energy transfer – Förster resonance energy transfer (BRET-FRET) technique is being established in living cells to establish close proximity of three proteins of interest. One protein is tagged with Renilla luciferase (Rluc) that activates the substrate Colentrazine H (ColH) and acts as a BRET donor (emitting 480 nm light). Another protein carries an EYFP fluorescent-tag, which is both a BRET acceptor and FRET donor (excited at 480 nm and emits at 525 nm). The third protein is tagged with the FRET acceptor (mOrange2 excited at 525 nm and emits at 565 nm). At 37°C, biolumiescence and fluorescence signals from living cells transfected with physiological quantities of these proteins are recorded in a cuvette by photomultiplier (PMT) based spectrometry and by microscopy using an electron multiplying charged coupled device (EM-CCD camera). Results: Fluorescence spectrometry of control Rluc-EYFP constructs transiently transfected into HEK cells was used to determine excitation and emission spectra. The excitation light was eliminated and the baseline bioluminescence and fluorescence signals (approximately 8 cps) were recorded without ColH. Robust increases in both signals (200 to 1000 fold) were observed immediately after addition of 5 mM ColH. Similar results were obtained at the single cell level by microscopy, with an observed 20-100 fold increase in EYFP signal after ColH addition. Conclusion: We have established a live cell microscopical BRET technique in the laboratory for the study of multiprotein complex dynamics.