Positron emission tomography (PET) and PET/computed tomography (PET-CT) studies with 11C- or 18F-labeled choline derivatives are used for PET imaging in glioblastoma patients. However, the nature of the choline transport system in glioblastoma is poorly understood. In this study, we performed a functional characterization of [methyl-3H]choline uptake and sought to identify the transporters that mediate choline uptake in the human glioblastoma cell lines A-172 and U-251MG. In addition, we examined the influence of anti-cancer drugs and central nervous system drugs on the transport of [methyl-3H]choline. High- and low-affinity choline transport systems were present in A-172 cells, U-251MG cells and astrocytes, and these were Na+-independent and pH-dependent. Cell viability in A-172 cells was not affected by choline deficiency. However, cell viability in U-251MG cells was significantly inhibited by choline deficiency. Both A-172 and U-251MG cells have two different choline transporters, choline transporter-like protein 1 (CTL1) and CTL2. In A-172 cells, CTL1 is predominantly expressed, whereas in U-251MG cells, CTL2 is predominantly expressed. Treatment with anti-cancer drugs such as cisplatin, etoposide and vincristine influenced [methyl-3H]choline uptake in U-251MG cells, but not A-172 cells. Central nervous system drugs such as imipramine, fluvoxamine, paroxetine, reboxetine, citalopram and donepezil did not affect cell viability or [methyl-3H]choline uptake. The data presented here suggest that CTL1 and CTL2 are functionally expressed in A-172 and U-251MG cells and are responsible for [methyl-3H]choline uptake that relies on a directed H+ gradient as a driving force. Furthermore, while anti-cancer drugs altered [methyl-3H]choline uptake, central nervous system drugs did not affect [methyl-3H]choline uptake.