The modem technique of photoacoustic calorimetry (PAC) has been critically evaluated for its utility and reliability in biophysical chemistry by applying it to reactive intermediates in the vitamin B-12 family of compounds. Measurement of the photoacoustic signals at 337 nm in 0.01 M phosphate buffer at pH 7.0, for methylcobalamin and methylcobinamide versus reference compounds from -2 to +40 degrees C, combined with measurement of the quantum yields for photodissociation (0.27 and 0.17, respectively), yields the bond dissociation energies of the corresponding cobalt-carbon bonds as BDE{Me-Cbl} = 36 and BDE{Me-Cbi} = 37 kcal mol(-1). Simultaneously, the reaction volumes for homolytic bond cleavage were determined to be 16 and 14 cm(3) mol(-1), respectively. These accurate measurements provide new paradigms for understanding the mechanisms by which the novel rearrangements catalyzed by coenzyme B-12 are achieved as well as establishing the validity of the PAC technique and data treatment. The further extension of PAC to the energetic analysis of the reactive intermediate methyl radical produced in the homolysis argues strongly for the broad utility of this systematic approach.