Carnosine (-alanyl-L-histidine) is present in high concentrations in human skeletal muscles. The oral ingestion of -alanine, the rate-limiting precursor in carnosine synthesis, has been shown to elevate the muscle carnosine content both in trained and untrained humans. Little human data exist about the dynamics of the muscle carnosine content, its metabolic regulation and its dependence upon muscle fiber type. The present study aimed to investigate in three skeletal muscle types the supplementation-induced amplitude of carnosine synthesis and its subsequent elimination upon cessation of supplementation (washout). Fifteen untrained males participated in a placebo-controlled double-blind study. They were supplemented for 5-6 weeks with either 4.8g/day -alanine or placebo. Muscle carnosine was quantified in soleus, tibialis anterior and medial head of the gastrocnemius by proton magnetic resonance spectroscopy (MRS), before and after supplementation and 3 and 9 weeks into washout. The -alanine supplementation significantly increased the carnosine content in soleus by 39%, in tibialis by 27% and in gastrocnemius by 23%, and declined post-supplementation at a rate of 2-4% per week. Muscle carnosine remained increased compared to baseline at 3 weeks of washout (only a third of the supplementation-induced increase had disappeared) and returned to baseline values within 9 weeks. Following subdivision into high-responders (+55%) and low-responders (+15%), washout period was 15 and 6 weeks, respectively.In the placebo group, carnosine remained relatively constant with variation coefficients of 9-15% over a 3-month period. It can be concluded that carnosine is a stable compound in human skeletal muscle, confirming the absence of carnosinase in myocytes. The current study shows that washout periods for cross-over designs in supplementation studies for muscle metabolites may sometimes require months rather than weeks.