We characterize the capabilities and limitations of the Living Image Software 3D Analysis package (Xenogen, Alameda, California) in the reconstruction of calibrated light sources. Sources shallower than the mean free path of light propagation suffered reconstruction inaccuracy. For sources deeper than the mean free path, the average error in depth and intensity reconstruction was less than 4% and 12%, respectively, for homogeneous tissue. The reconstruction of luminescent beads implanted within an optically heterogeneous mouse abdomen proved less accurate. The ability to distinguish multiple sources decreased with increasing source depth. A number of factors influence the accuracy of light source reconstruction.