Blood perfusion in organs has been shown to be heterogeneous in a number of cases. At the same time, a number of models of vascular structure and flow have been proposed that also generate heterogeneous perfusion. While a relationship between local perfusion and vascular structure has to exist, no model has yet been validated as an accurate description of this relationship. A study of perfusion and 3D arterial structure in individual rat kidneys is presented, which allows comparison between local measurements of perfusion and model-based predictions. High-resolution computed tomography is used to obtain images of both deposited microspheres and of an arterial cast in the same organ. Microsphere deposition is used as an estimate of local perfusion. A three-dimensional cylindrical pipe model of the arterial tree is generated based on an image of the arterial cast. Results of a flow model are compared with local microsphere deposition. High correlation (R² > 0.94) was observed between measured and modeled flows through the vascular tree segments. However, the relative dispersion (RD) of the microsphere perfusion measurement was 2- to 3-fold higher than perfusion heterogeneity calculated in the flow model. Also, there was no correlation in the residual deviations between the methods. This study illustrates the importance of comparing models of local perfusion with in vivo measurements of perfusion in the same biologically realistic vascular tree.