Glutamate is the major excitatory neurotransmitter in the brain. Glutamate activation of the N-methyl-D-aspartate (NMDA) receptor subtype is thought to mediate important physiological and pathological processes, including memory formation and excitotoxicity. The goal of the present work was to characterize and validate a candidate agent for noninvasive positron emission tomography (PET) imaging of this receptor. [(3)H]-labeled N-[3-(3)H]-methyl-3-(thiomethylphenyl)cyanamide (CNS-5161) was incubated with rat brain homogenates at increasing concentrations, temperatures, and times to establish the binding kinetics and affinity of the ligand in vitro. Nonspecific binding was measured with 100 microM MK-801. The compound was also injected i.v. in rats pretreated with saline, NMDA, MK801, or a combination, and organ and brain regional uptake was assessed at various times after injection by autoradiography or dissection. Blood and brain samples were assayed for metabolites by high-performance liquid chromatography. CNS-5161 binds brain membranes with high affinity (K(d) < 4 nM) and fast association and dissociation kinetics. Specific binding increased in the presence of glutamate and glycine. Intravenous administration in control rats resulted in a heterogeneous brain distribution with hippocampus and cortex > thalamus > striatum > cerebellum, and a cortex/cerebellum ratio of 1.4. Pretreatment with NMDA increased the hippocampus-to-cerebellum ratio to 1.6-1.9 while MK801 abolished this increase, resulting in ratios close to 1. Thus, CNS-5161 binds preferentially to the activated state of the NMDA receptor channel in vitro and in vivo. The high affinity and fast kinetics make it compatible with PET imaging of a carbon-11 labeled CNS-5161.