To test the hypothesis that creatine supplementation would enhance the anabolic responses of muscle cell signalling and gene expression to exercise, we studied nine subjects who received either creatine or a placebo (maltodextrin) for 5d in a double-blind fashion before undergoing muscle biopsies: at rest; immediately after exercise (10x10 repetitions of one leg-extension at 80% 1-RM); and 24h and 72h later (all in the morning after fasting overnight). Creatine supplementation decreased the phosphorylation state of protein kinase B (PKB) on Thr308 at rest by 60% (P<0.05) and that of eukaryotic initiation factor 4E-binding protein on Thr37/46 (4E-BP1) by 30% 24h post-exercise (P<0.05). Creatine increased mRNA for collagen 1(1), glucose transporter-4 (GLUT-4) and myosin heavy chain I at rest by 250%, 45% and 80% respectively, and myosin heavy chain IIA (MHCIIA) mRNA immediately after exercise by 70% (all P<0.05). Immediately after exercise, and independent of creatine, mRNA for muscle atrophy F-box (MAFbx), MHCIIA, peroxisome proliferator-activated receptor coactivator-1 and interleukin-6 were up-regulated (60-350%, P<0.05); the phosphorylation state of p38 both in the sarcoplasm and nucleus were increased (12 and 25 fold respectively, both P<0.05). Concurrently, the phosphorylation states of PKB (Thr308) and 4E-BP1 (Thr37/46) were decreased by 50% and 75% respectively (P<0.05). Twenty-four hours post-exercise, MAFbx, myostatin and GLUT-4 mRNA expression decreased below pre-exercise values (-35 to -50%, P<0.05); calpain 1 mRNA increased 70% 72h post-exercise (P<0.05) and at no other time. In conclusion, 5d of creatine supplementation does not enhance anabolic signalling but increases the expression of certain targeted genes.