Spectrally resolved bioluminescence optical tomography is an approach to recover images of luciferase activity within a volume using multiwavelength emission data from internal bioluminescence sources. The underlying problem of uniqueness associated with nonspectrally resolved intensity-based bioluminescence tomography is highlighted. Reconstructed images of bioluminescence are presented by using as input both simulated and real multiwavelength data from a tissue-simulating phantom. The location of the internal bioluminescence is obtained with 1 mm accuracy. Further, the amplitude of the reconstructed source is proportional to the actual bioluminescence intensity.