[99mTc]HM-PAO exists as two stereoisomers, d,1 and meso, only one of which is retained in the brain. It has been suggested recently that the biodistribution of [99mTc]d,1-HM-PAO can be explained by its interaction with glutathione (GSH) in the tissues. We studied the interactions of the d,1 and meso isomers with GSH in vitro by measuring the partitioning of activity between ethyl acetate and aqueous GSH solutions at various concentrations. Partitioning of both isomers demonstrated a sigmoidal relationship with GSH concentration, but the d,1 isomer showed eightfold greater reactivity than the meso isomer. In a separate experiment, the d,1 isomer showed a sevenfold greater interaction rate with GSH than the meso isomer. These results suggest that the stereoisomers of [99mTC]HM-PAO show differences in their interaction rate with GSH which may explain their different retention in the brain.