2 beta-Carbomethoxy-3 beta-(4-iodophenyl)tropane (beta-CIT; also designated RTI-55) is an analog of cocaine that has been developed as a single photon emission computed tomography radiotracer that labels dopamine and serotonin transporters. We have prepared the 125I- and 123I-labeled ([1R] "active" and [1S] "inactive") enantiomers of beta-CIT. Total homogenate binding of the 125I-labeled inactive isomer to baboon caudate and cortex was approximately equal to nonspecific binding of the active isomer in cortex and much lower than total binding of the active isomer in caudate. However, inactive isomer homogenate binding in caudate was somewhat higher than in cortex, and during single photon emission computed tomography scanning in vivo striatal (1S)-[123I]beta-CIT uptake was also slightly greater than in cortex. Following intravenous administration of the 123I-labeled enantiomers, the plasma clearances of the active and inactive enantiomers were not significantly different. Single photon emission computed tomography imaging demonstrated that a bolus dose of nonradioactive (1R)-beta-CIT rapidly displaced the uptake of (1R)-[123I]beta-CIT. In contrast, the brain uptake of (1S)-[123I]beta-CIT was not displaced by nonradioactive (1R)-beta-CIT using either a bolus ("kinetic") or bolus plus constant infusion ("equilibrium") paradigm for administration of the radiotracer. In scans with bolus administration of radiotracer, peak striatal uptake of the active isomer was approximately twice that of the inactive isomer. In comparison to the 123I-labeled active tracer, the inactive tracer showed earlier times to peak activity and faster washouts of activity in all brain regions. These studies demonstrate beta-CIT stereoselectivity using both homogenate binding and in vivo imaging and suggest that the inactive enantiomer may be a useful measure of the kinetics of both blood-brain barrier transport and nonspecific binding.