The betaine/γ-aminobutyric acid (GABA) transporter 1 (BGT1) is one of the four GABA transporters (GATs) involved in the termination of GABAergic neurotransmission. Although suggested to be implicated in seizure management, the exact functional importance of BGT1 in the brain is still elusive. This is partly owing to the lack of potent and selective pharmacological tool compounds that can be used to probe its function. We previously reported the identification of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA), a selective substrate for BGT1 over GAT1/GAT3, but also an agonist for GABAA receptors. With the aim of providing new functional insight into BGT1, we here present the synthesis and pharmacological characterization of the tritiated analogue, [(3)H]ATPCA. Using traditional uptake assays at recombinant transporters expressed in cell lines, [(3)HH]ATPCA displayed a striking selectivity for BGT1 among the four GATs (Km and Vmax values of 21 µM and 3.6 nmol ATPCA/(min×mg protein), respectively), but was also found to be a substrate for the creatine transporter (CreaT). In experiments with mouse cortical cell cultures, we observed a Na(+)H-dependent [(3)HH]ATPCA uptake in neurons, but not in astrocytes. The neuronal uptake could be inhibited by GABA, ATPCA and a non-competitive BGT1-selective inhibitor, indicating functional BGT1 in neurons. In conclusion, we report [(3)HH]ATPCA as a novel radioactive substrate for both BGT1 and CreaT. The dual activity should be taken into consideration when using this tool compound to study either target under native conditions.