Proteasome inhibitors are novel potential drugs for therapy of many diseases, and their effects are not fully understood. We investigated direct effects of peptide vinylsulfone inhibitor AdaAhx3L3VS on protein and amino acids metabolism in rat skeletal muscle. Soleus and extensor digitorum longus muscles were incubated in a medium containing 30 micromol/l AdaAhx3L3VS or no inhibitors. Total proteolysis was determined according to the rates of tyrosine release into the medium during incubation. The rates of leucine oxidation and protein synthesis were evaluated during incubation in medium containing L-[1-14C]leucine. Amino acid concentrations in the medium were measured using HPLC. AdaAhx3L3VS decreased tyrosine release into the medium by 21 and 19 %, decreased leucine incorporation into proteins by 22 and 12 %, and increased leucine oxidation by 24 and 19 % in soleus and extensor digitorum longus muscles, respectively. The release of amino acids into the medium was reduced. We conclude that AdaAhx3L3VS significantly decreased proteolysis and protein synthesis and increased leucine oxidation.