The purpose of this study was to discern the critical point in skeletal muscle fatty acid oxidation by changing plasma free fatty acids (FFA) level in rat. In the study, 3 key steps in lipid oxidation were examined after changing plasma FFA level by acipimox. The rates of both palmitate and palmitoyl- carnitine oxidation were decreased by decrease of plasma FFA level, however, carnitine palmitoyl transferase (CPT) 1 activity was not changed, suggesting CPT1 activity may not be involved in the fatty acid oxidation at the early phase of plasma FFA change. In the fasted rats, β-hydroxy acyl-CoA dehydrogenase (β-HAD) activity was depressed to a similar extent as palmitate oxidation by a decrease of plasma FFA level. This suggested that β-oxidation might be an important process to regulate fatty acid oxidation at the early period of plasma FFA change. Citrate synthase activity was not altered by the change of plasma FFA level. In conclusion, the critical step in fatty acids oxidation of skeletal muscles by the change of plasma FFA level by acipimox in fasting rats might be the β-oxidation step rather than CPT1 and TCA cycle pathways.