The oxidation enthalpy of reduced flavin mononucleotide at pH 7.0 in 0.2 m phosphate buffer has been studied by determining the heat associated with the reaction: FMNH 2 + 2 Fe(CN) −3 6 ⇌ FMN + 2 Fe(CN) −4 6 + 2 H +. (a) (The quinone, semiquinone, and hydroquinone forms of FMN are represented as FMN, FMNH, and FMNH 2, respectively.) Calorimetric experiments were performed in a flow microcalorimeter which was modified to prevent sample contamination by oxygen. The enthalpy observed for reaction (a), after correction for dilution and buffer effects, was −39.2 ± 0.4 kcal (mole FMNH 2) −1 at 25 °C. The potential difference, ΔE′, developed by reaction (a) was determined potentiometrically and corresponded to a free energy change, ΔG′, of −30.3 kcal (mole FMNH 2) −1. The resulting entropy change, ΔS′, was thus calculated to be −29.8 e.u. Reaction (a) was also studied at temperatures of 7 °C and 35.5 °C. ΔC p ′ for the reaction was calculated as −155 ± 18 cal deg −1 (mole FMNH 2) −1 at 20 °C. ΔH′ for the reaction (b), FMNH 2 ⇌ FMN + H 2, (b) was calculated as +14.2 ± 0.7 kcal mole −1 at 25 °C, relative to the enthalpy of the hydrogen electrode being identically equal to zero at all values of pH and temperature. The free energy at pH 7.0 for reaction (b), calculated from the potential was found to be −9.7 kcal mole −1, which resulted in an entropy for reaction (b) of 80.2 e.u. A thermal titration of reaction (a) was used to calculate the thermodynamic parameters for the formation of semiquinone dimer according to the reaction FMNH 2 + FMN ⇌ (·FMNH) 2. (c) The free energy, enthalpy, and entropy changes for reaction (c) were estimated to be −6.1 kcal mole −1, −7 kcal mole −1, and −3 e.u., respectively.