SPECT imaging of benzodiazepine (BZ) receptor using the antagonist [123I]iomazenil in nonhuman primates was correlated with ex vivo autoradiography and in vitro receptor autoradiography of postmortem tissue sections taken at the time of maximal brain uptake of the radiotracer. Cryostat sections prepared from brain tissue blocks at the orientation of the SPECT imaging plane showed high activity in gray matter regions, with gray to white matter ratios of greater than 30:1. After at least one week decay of 123I, these same tissue sections were used to localize and quantify the distribution of BZ receptors with the standard technique of in vitro receptor autoradiography using [125I]iomazenil. The ex vivo autoradiographic distribution of activity was highly correlated (r = 0.89) with the distribution of BZ receptors, although a few brain regions showed reproducible discrepancies between ex vivo and in vitro results. The ex vivo autoradiograms provided quantitative data from a realistic "biological phantom," which may be used to assess the accuracy of image reconstruction and to investigate differences between the distribution of an intravenously (i.v.) administered tracer and that of its target in brain tissue.