Spectrally resolved bioluminescence optical tomography is an approach to recover images of, for example, 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 demonstrated and it is shown that using a non-negative constraint inverse algorithm, an accurate solution for the source distribution can be calculated from the measured data. Reconstructed images of bioluminescence are presented using both simulated complex and heterogeneous small animal models as well as real multiwavelength data from a tissue-simulating phantom. The location of the internal bioluminescence source using experimental data is obtained with accuracy and it is shown that small ( diameter) sources of up to deep, within a complex mouse model, can be resolved accurately using a single view data collection strategy. Finally, using the reciprocity approach for image reconstruction, a dramatic improvement in computational time is shown without loss to image accuracy with both experimental and simulated data, potentially reducing computing time from .