We report the use of a continuous-wave (CW) CO2 laser for the determination of relative activation energy for unimolecular dissociation of large biomolecular ions. The [M + 5H](5+) and [M + 11H](11+) ions of bovine ubiquitin and the [M + H](+) ion of bradykinin are irradiated with a CW CO2 laser and the rate constant for dissociation at each of several laser intensities recorded. A plot of the natural logarithm of the first-order rate constant versus the natural logarithm of laser intensity yields a straight line whose slope provides an approximate measure of the activation energy (E-a) for dissociation. For dissociation of protonated bradykinin, the absolute E-a value from infrared multiphoton dissociation (IRMPD) agrees with that obtained by blackbody infrared radiative dissociation (BIRD), whereas the IRMPD-determined E(a)s for dissociation of the 5+ and 11+ charge states of bovine ubiquitin are lower than those obtained by BIRD. The relative E-a values for the 5+ and 11+ charge states of bovine ubiquitin from both BIRD and IRMPD are in good agreement. Master equation modeling was carried out on the model peptide, (AlaGly)(8), to characterize the nature of the internal energy distribution produced from irradiation by a monochromatic IR source (e.g., CW CO2 laser) versus a broadband IR source (e.g., blackbody). The master equation simulation shows that the internal energy distribution produced by irradiation with the CO2 laser is essentially identical to that obtained by blackbody irradiation. Our combined experimental and theoretical results justify the IRMPD technique as a viable method for the determination of relative ordering of activation energies for dissociation of large (>50 atoms) ions.