Our aim was to determine the disposition of creatine in ovine pregnancy, and whether creatine is transferred across the placenta from mother to fetus. Pregnant ewes received either: (i) a continuous intravenous infusion of creatine monohydrate or saline from 122 to 131 days gestation, with maternal and fetal arterial blood and amniotic fluid samples collected daily for creatine analysis and fetal tissues collected at necropsy at 133 days for analysis of creatine content, or; (ii) a single systemic bolus injection of ¹³C labelled creatine monohydrate at 130 days of gestation, with maternal and fetal arterial blood, uterine vein blood and amniotic fluid samples collected prior to, and for 4 hours post injection and analysed for creatine, creatine isotopic enrichment, and guanidinoacetic acid (GAA, precursor of creatine) concentrations. Presence of the creatine transporter-1 (SLC6A8), and arginine-glycine amidinotransferase (AGAT, the enzyme synthesising GAA) proteins were determined by western blots of placental cotyledons. The 10-day creatine infusion increased maternal plasma creatine concentration 3-4 fold (P<0.05), without significantly changing fetal arterial, amniotic fluid, fetal tissues, or placental creatine content. Maternal arterial ¹³C enrichment was increased (P<0.05) post bolus ¹³C-creatine injection, without change of fetal arterial ¹³C enrichment. SLC6A8 and AGAT proteins were identified in placental cotyledons, and GAA concentration was significantly higher in uterine vein than maternal artery plasma. Despite the presence of SLC6A8 protein in cotyledons, these results suggest that creatine is not transferred from mother to fetus in near-term sheep, and that the ovine utero-placental unit releases GAA into the maternal circulation.