Although creatine is one of the most widely used nutritional supplements for athletes as well as for patients with neuromuscular disorders, the effects of oral creatine supplementation on endogenous creatine synthesis in humans remains largely unexplored. The aim of the present study was to investigate the metabolic consequences of a frequently used long-term creatine ingestion protocol on the circulating creatine synthesis precursor molecules, guanidinoacetate and arginine, and their related guanidino compounds. For this purpose, sixteen healthy young volunteers were randomly divided to ingest in a double-blind fashion either creatine monohydrate or placebo (maltodextrine) at a dosage of 20g/day for the first week (loading phase) and 5g/day for 19 subsequent weeks (maintenance phase). Fasting plasma samples were taken at baseline and at 1, 10 and 20 weeks of supplementation and guanidino compounds were determined. Plasma guanidinoacetate levels were reduced by 50% after creatine loading and remained ~30% reduced throughout the maintenance phase. Several circulating guanidino compound levels were significantly altered after creatine loading but not during the maintenance phase: homoarginine (+35%), -keto--guanidinovaleric acid (+45%) and argininic acid (+75%) were increased, whereas guanidinosuccinate was reduced (-25%). The decrease in circulating guanidinoacetate levels suggests that exogenous supply of creatine chronically inhibits endogenous synthesis at the transamidinase step in humans, supporting earlier animal studies showing a powerful repressive effect of creatine on arginine:glycine amidinotransferase (AGAT). Furthermore, these data suggest that this leads to enhanced utilization of arginine as a substrate for secondary pathways.