Technetium-99m-labeled radiopharmaceuticals are currently the most commonly used agents in nuclear medicine. To prepare binding site-specific small molecules containing a Tc-99m complexing core, it is important to consider a ligand system, which selectively forms only one stereoisomer. A novel series of bisaminoethanethiol (BAT) derivatives as a model system were prepared. Stereoisomers of N-benzyl-3,4-di(N-2-mercaptoethyl)-amino pyrrolidines (P-BAT): (3R,4R)-P-BAT (R,R-4) and (3,4)meso-P-BAT (8), the trans and meso isomer, respectively, as a chelating group were prepared successfully. The desired Tc-99m P-BAT complexes were obtained by using Sn(II)/glucoheptonate as the reducing agent for [99mTc]pertechnetate. As predicted, after complexation with [99mTc]Tc'O, the trans isomer, (3R,4R)-P-BAT (R,R-4), showed only one isomer; whereas the corresponding meso isomer, (3,4)meso-P-BAT (8), produced two distinctive complexes isolated readily by high performance liquid chromatography (HPLC). The [99mTc](R,S) meso-P-BAT (8) isomers showed a different lipophilicity (partition coefficient [P.C.] = 54.3 and 55.4 for peak A and peak B, respectively), as compared with that of the corresponding [99mTc](3R,4R)-P-BAT (R,R-4), trans isomer (P.C. = 163). Results of the biodistribution study in rats of these isomers show different heart and brain uptake, suggesting that the intrinsic differences in biodistribution are due to structural and stereospecific factors. Examples in this report confirm that it is possible to design stereospecific Tc-99m complexes based on the bisaminoethanethiol (N2S2, BAT) ligand system. Consideration on stereoselectivity of site-specific agents labeled with Tc-99m is likely an essential requirement on developing binding-site specific radiopharmaceuticals.