At excitation wavelengths in the extreme red onset region of the trans,trans-1,4-diphenyl-1,3-butadiene (DPB) absorption spectrum, observed fluorescence spectra are mixtures of contributions from s-trans and s-cis conformers. Quantitative separations of the spectral mixtures by use of a chemometric method make it possible to determine relative fluorescence contributions and fluorescence quantum yields of DPB conformers as a function of temperature. Enthalpy differences of 1.9 and 2.3 kcal/mol are obtained for the ground state conformational equilibrium of DPB in methylcyclohexane and toluene, respectively. In computational studies, conformational energies and geometries of DPB are evaluated in terms of an ab initio method. The calculated s-cis-s-trans energy difference is in surprisingly good agreement with the experimental results.