The present study aimed to investigate whether skeletal muscle from whole body creatine transporter (CrT; SLC6A8) knockout mice (CrT-/y) actually contained creatine (Cr) and if so, whether this Cr could result from an up regulation of muscle Cr biosynthesis. Gastrocnemius muscle from CrT-/y and wild type (CrT+/y) mice were analysed for ATP, Cr, Cr phosphate (CrP) and total Cr (TCr) content. Muscle protein and gene expression of the enzymes responsible for Cr biosynthesis L-arginine:glycine amidotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) were also determined as were the rates of in vitro Cr biosynthesis. CrT-/y mice muscle contained measurable (22.3 ± 4.3 mmol.kg-1 dry mass), but markedly reduced (P<0.05) TCr levels compared with CrT+/y mice (125.0 ± 3.3 mmol.kg-1 dry mass). AGAT gene and protein expression were higher (~3 fold; P<0.05) in CrT-/y mice muscle, however GAMT gene and protein expression remained unchanged. The in vitro rate of Cr biosynthesis was elevated 1.5 fold (P<0.05) in CrT-/y mice muscle. These data clearly demonstrate that in the absence of CrT protein, skeletal muscle has reduced, but not absent, levels of Cr. This presence of Cr was most likely due to an up regulation of muscle Cr biosynthesis as evidenced by an increased AGAT protein expression and in vitro Cr biosynthesis rates in CrT-/y mice. Of note, the up regulation of Cr biosynthesis in CrT-/y mice muscle was unable to fully restore Cr levels to that found in wild type muscle.