C6 glioma cells have been proposed as a cell model system to study regulation of endogenously-expressed glycine transporter 1 (GlyT1). However, there are no reports demonstrating expression of glycine transporter 2 (GlyT2) in this cell line. To explore this possibility, immunoprecipitation, western blotting, confocal microscopy, as well as glycine uptake assays using selective inhibitors to GlyT1 or GlyT2 were employed. Glycine uptake assays using a selective inhibitor against GlyT2 provided evidence that this transporter could also be expressed in this cell line. Immunoprecipitation, immunoblotting, and confocal microscopic analysis also showed presence of both GlyT1 and GlyT2 in this cell line. Taken together, results provide evidence that in addition to GlyT1, GlyT2 is also endogenously expressed in C6 glioma cells. Results presented here will help researchers to carefully evaluate use of this cell line in studies involving GlyT1.