In developing new brain perfusion imaging agents, we prepared 99mTc complexes of racemic mixtures of bis(aminoethanethiol) (BAT) derivatives containing an N'-benzylpiperazinyl (BPA) side chain. Due to the presence of a chiral center, a mixture of diastereomers (syn and anti) following chelation with the 99mTc (no-carrier-added) was obtained. The neutral and lipid-soluble 99mTc-BPA-BAT (99mTc, T1/2 = 6 h) isomers were separated. The syn and anti isomers of carrier-added 99Tc-BPA-BAT (99Tc, T1/2 = 2 x 10(5) years) were also synthesized, separated, and crystallized. The X-ray crystallography of 99Tc-BPA-BAT showed the syn and anti conformations (in relationship with the central TC(=O)N2S2 core). Despite a similarity in the partition coefficients for the two isomers, the syn isomer showed a higher in vivo brain uptake and longer brain retention in rats (2.77 and 1.08% dose/organ at 2 and 15 min) than that of the corresponding anti isomer (0.57 and 0.27% dose/organ at 2 and 15 min). This information is important and should be taken into consideration when new 99mTc-labeled brain perfusion imaging agents are being designed.