Introduction Recently, we have reported modification of 99mTc-TRODAT-1 by integrating the N2S2 metal chelating unit and the tropane skeleton. Results of a preliminary biodistribution study in rats were promising with respect to brain uptake. The present report deals with the further biological characterization of the 99mTc-labelled integrated TRODAT derivatives ( 99mTc-TropaBAT and 99mTc-norchloro-TropaBAT) and with the synthesis and biological evaluation of a novel 99mTc-labelled piperidine-based derivative ( 99mTc-PipBAT). Methods Biodistribution of all radiolabelled complexes was studied in normal mice. A more detailed ex vivo intracerebral distribution study of the two 99mTc-TropaBAT complexes was additionally performed in normal rats. Autoradiography of brain sections of normal mice (with or without pretreatment with FP-β-CIT or haloperidol) and rats was performed. Affinity for the dopamine transporter (DAT) was also assessed in vitro in the presence or absence of cocaine. Results Both 99mTc-TropaBAT complexes show a slightly higher brain uptake than 99mTc-TRODAT-1, but the striatum/cerebellum activity ratio is less favourable. Nevertheless, significant striatal uptake was detected after ex vivo autoradiography, but this uptake was also observed after pretreatment with FP-β-CIT. Unexpectedly, no striatal uptake was detected after in vitro incubation of mouse brain sections with the tracer agents. For 99mTc-PipBAT, neither brain uptake nor in vitro striatal uptake was found. Conclusion Both 99mTc-TropaBAT complexes exhibit similar diffusion into brain as 99mTc-TRODAT-1, and ex vivo autoradiography shows significant striatal uptake. However, the inferior striatum/cerebellum activity ratio, the striatal uptake in mice pretreated with FP-β-CIT or haloperidol, and the lack of striatal uptake during in vitro incubation prove that the DAT is not targeted. Brain uptake disappears when the tropane skeleton is replaced by a piperidine ring, and also in this case no striatal uptake is found in vitro.