In this study, we demonstrate two new concepts, using fluorescence correlation spectroscopy (FCS), to characterize fluorescence resonance energy transfer (FRET). The two approaches were tested experimentally by measuring a series of double-stranded DNA molecules, with different numbers of base-pairs separating the donor (Alexa488) and acceptor (Cy5) fluorophores. In the first approach, FRET efficiencies are determined from the detected acceptor fluorescence rate per molecule. Here, the unique possibility with FCS to determine the mean number of molecules within the detection volume is exploited, making a concentration calibration superfluous. The second approach takes advantage of FRET-dependent fluorescence fluctuations of photophysical origin, in particular fluctuations generated by trans−cis isomerization of the acceptor dye. The rate of interchange between the trans and cis states is proportional to the excitation rate and can be conveniently measured by FCS. Under FRET-mediated excitation, this rate ...