Purpose: To test the use of a three-dimensional (3D) optical cone beam computed tomography reconstruction algorithm, for estimation of the imparted 3D dose distribution from megavoltage photon beams in a water tank for quality assurance, by imaging the induced Cherenkov-excited fluorescence (CEF). Methods: An intensified charge-coupled device coupled to a standard nontelecentric camera lens was used to tomographically acquire two-dimensional (2D) projection images of CEF from a complex multileaf collimator (MLC) shaped 6 MV linear accelerator x-ray photon beam operating at a dose rate of 600 MU/min. The resulting projections were used to reconstruct the 3D CEF light distribution, a potential surrogate of imparted dose, using a Feldkamp-Davis-Kress cone beam back reconstruction algorithm. Finally, the reconstructed light distributions were compared to the expected dose values from one-dimensional diode scans, 2D film measurements, and the 3D distribution generated from the clinical Varian ECLIPSE treatment planning system using a gamma index analysis. A Monte Carlo derived correction was applied to the Cherenkov reconstructions to account for beam hardening artifacts. Results: 3D light volumes were successfully reconstructed over a 400 × 400 × 350 mm 3 volume at a resolution of 1 mm. The Cherenkov reconstructions showed agreement with all comparative methods and were also able to recover both inter-and intra-MLC leaf leakage. Based upon a 3%/3 mm criterion, the experimental Cherenkov light measurements showed an 83%-99% pass fraction depending on the chosen threshold dose. Conclusions: The results from this study demonstrate the use of optical cone beam computed tomography using CEF for the profiling of the imparted dose distribution from large area megavoltage photon beams in water. C 2015 American Association of Physicists in Medicine.