Six enantiomers of three racemic sigma-1 receptor (σR) ligands were resolved, and absolute configuration was determined. Their high σR potency and selectivity were determined through in vitro binding assays, further validated by molecular docking analysis. Central Nervous System Multiparameter Optimization algorithm (CNS MPO) predicts efficient brain penetration for these enantiomers. Six C-11 radiotracers were radiosynthesized successfully, ex vivo biodistribution in rats showed that (-)-[C]7 had high brain uptake of ∼4.8-fold for 5 min versus 60 min. Mouse brain PET imaging studies showed (-)-[C]7 and (-)-[C]16 have in vivo binding specificity for σR. Macaque PET scans showed high brain uptake for all six radiotracers, with (-)-[C]7 peaked at ∼45 min (SUV 2.5), possessing the best washout kinetics and highest cerebellum-to-white matter ratio (∼3.1), in agreement with in vitro or ex vivo measures of σR expression. Radiometabolite analysis showed that no newly formed radiometabolite was observed post-injection of (-)-[C]7. Our data suggest that further evaluation is warranted to determine that (-)-[C]7 is a suitable PET radiotracer for imaging σR in the brain of animal and human.