The effect of increased free fatty acid concentrations on glucose metabolism in rat skeletal muscle was investigated at several different steps in glucose metabolism including glucose transport, glucose phosphorylation, glucose oxidation and glycogen synthesis. In isolated soleus (slow-twitch) muscles, insulin-stimulated (100 microU ml-1) glucose phosphorylation, but not glucose transport, was inhibited by 26 and 22% in the presence of 1.0 and 2.0 mM oleate, respectively (P < 0.01). Regardless of oleate concentration (0.3 or 2.0 mM), insulin-stimulated glucose 6-phosphate levels were elevated to the same extent over the non-insulin-stimulated levels in soleus muscles (P < 0.01). Insulin-stimulated glucose oxidation was inhibited by 44% in soleus muscles exposed to 2.0 mM oleate (P < 0.05), whereas the rate of glucose incorporation into glycogen was not altered. In insulin-stimulated epitrochlearis (fast-twitch) muscles, elevated concentrations of oleate had no effect on the rates of glucose transport or glucose phosphorylation, or on the level of glucose 6-phosphate. These data suggest that increased free fatty acid availability decreases glucose utilization by selectively inhibiting glucose phosphorylation and oxidation in slow-twitch, but not fast-twitch skeletal muscle.