Oral creatine supplementation may be useful to prevent and/or treat neurodegenerative pathologies. Creatine transporter (CRT) is widely distributed in the central nervous system and it has been functionally characterized in cells transfected with CRT cDNA. However, information regarding brain creatine transport is lacking. In the present study we investigate the transport of creatine in synaptosomes isolated from the rat telencephalum, diencephalum and cerebellum, and its variation with age. The experiments were performed in accordance with the current national and local guidelines for the care and use of laboratory animals. Six Wistar rats of either 18-day (suckling) or 2-month-old (adult) were anaesthetized with ether and then humanely killed. Whole brains were removed and crude synaptosomal fractions of telencephalum and diencephalum were prepared using ficoll gradient (1). [¹⁴C]-creatine uptake was measured in brain synaptosomal preparations using a rapid filtration technique (2) and expressed as pmol/mg protein/3 min. ANOVA followed by Newman-Keuls’ test was used for statistical analysis. Brain synaptosomes accumulate creatine against its concentration gradient. This accumulation was Na⁺-dependent (5.0 ± 0.4 and 7.2 ± 0.6 in the telencephalum, and 12.3 ± 1.3 and 14.0 ± 1.5 in the diencephalum, of suckling and adult rats, respectively) and antagonized by the specific creatine transport inhibitor guanidinopropionic acid (2.66 ± 0.24, p<0.001 as compared with Na⁺-dependent creatine uptake). Na⁺-independent creatine uptake ranged from 1.9 ± 0.4 to 2.6 ± 1.3. In both suckling and adult rats, creatine transport activity was lower in the telencephalum than in the diencephalum (p<0.001), and it was significantly (p<0.05) increased with age in the telencephalum. Northern assays revealed two CRT mRNA transcripts, of 4.2-kb and 2.7-kb in the brain isolated from suckling rats. The expression of the 4.2-kb transcript was 5 to 6 times higher than that of the 2.7-Kb transcript and it was significantly (p<0.05) less abundant in the telencephalum than in the other brain regions examined. The 2.7-Kb transcript was equally distributed in the three brain regions examined. The Western blots revealed immunoreactive bands of 57-, 60- and 71-kDa in the three brain regions studied. In suckling rats, the three proteins were more abundant in the diencephalum and cerebellum than in the telencephalum. In adult rats, however, only the 71-kDa protein was more abundant in the diencephalum and cerebellum than in the telencephalum, whereas the abundance of the other two proteins were lower in the diencephalum and cerebellum than in the telencephalum. In conclusion, creatine transport in the brain appears to be mediated by the 71-kDa protein.