Single molecule fluorescence imaging has been used to unequivocally differentiate between rhodamine-6G labeled gramicidin monomeric subunits and channel-forming dimers. Absolute identification of individual particles was achieved by accounting for both particle diffusion and intensity, with dimer intensity being twice that of the monomers. In accordance with current diffusion models of proteins in bilayer membranes, we observed dimers to diffuse more slowly through the bilayer than the monomers and have reported diffusion coefficients of 1.2 x 10(-8) and 3.5 x 10(-8) cm(2)/s for the dimers and monomers, respectively. By correlating the diffusion data with measured fluorescence intensities of the tracked particles, it was possible to determine the distribution of monomers and dimers within the bilayer at various temperatures. The results allow complete characterization of the thermodynamic properties of dimer formation, 2G(1) <----> G(2), necessary for channel function. Reported are the temperature-dependent equilibrium constants, DeltaH(Reaction)degrees, DeltaG(Reaction)degrees, DeltaS(Reaction)degrees, formation in an artificial lipid membrane that has a thickness (30 Angstrom) which is on the same order as the length of the gramicidin channel (26 Angstrom). These experiments compliment and expand single molecule fluorescence methods needed to understand the complexities of ion channel structure/function relationships.