The flumazenil analogue, Ro 16-0154, a benzodiazepine partial inverse agonist, has been labeled by halogen exchange to enable SPECT investigations of central benzodiazepine receptors in the human brain. The purified 123I-Ro 16-0154 was found to be stable in rat brain preparations and to be metabolized in rat liver preparations. Its pharmacologic properties were comparable to those of flumazenil. The biodistribution in rats (1 hr postinjection) resulted in a high brain-to-blood ratio of 16. Clinical studies revealed images of the benzodiazepine receptor density in the brain. Since the receptor labeling was markedly reduced by injection of flumazenil, it was considered to be specific. Storage defects due to pathologic cerebral blood flow and changed receptor density were detected; this shows the potential usefulness of the substance for diagnostic purposes, e.g., the differential diagnosis of various forms of epilepsy.