Monte Carlo simulations used to determine the relationship between average FRET efficiency and Separation under static isotropic conditions. The probability distribution of FRET efficiencies derived from Monte Carlo simulations where separation, normalized to the Förster distance (Φ), was set to values of 0.66 (A), 0.89 (B), and 1.14 (C), corresponding to average population FRET efficiencies, 〈E〉, of approximately 75%, 50%, and 25%, respectively. Note the irregular shapes of all three populations.