Previous studies have shown that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a cell-permeable activator of AMP-activated protein kinase, increases the rate of fatty acid oxidation in skeletal muscle of fed rats. The present study investigated the mechanism by which this occurs and, in particular, whether changes in the activity of malonyl-CoA decarboxylase (MCD) and the β-isoform of acetyl-CoA carboxylase (ACCβ) are involved. In addition, the relationship between changes in fatty acid oxidation induced by AICAR and its effects on glucose uptake and metabolism was examined. In incubated soleus muscles isolated from fed rats, AICAR (2 mM) increased fatty acid oxidation (90%) and decreased ACCβ activity (40%) and malonyl-CoA concentration (50%); however, MCD activity was not significantly altered. In soleus muscles from overnight-fasted rats, AICAR decreased ACCβ activity (40%), as it did in fed rats; however, it had no effect on the already high rate of fatty acid oxidation or the low malonyl-CoA concentration. In keeping with its effect on fatty acid oxidation, AICAR decreased glucose oxidation by 44% in fed rats but did not decrease glucose oxidation in fasted rats. It had no effect on glucose oxidation when fatty acid oxidation was inhibited by 2-bromopalmitate. Surprisingly, AICAR did not significantly increase glucose uptake or assayable AMP-activated protein kinase activity in incubated soleus muscles from fed or fasted rats. These results indicate that, in incubated rat soleus muscle,1) AICAR does not activate MCD or stimulate glucose uptake as it does in extensor digitorum longus and epitrochlearis muscles,2) the ability of AICAR to increase fatty acid oxidation and diminish glucose oxidation and malonyl-CoA concentration is dependent on the nutritional status of the rat, and 3) the ability of AICAR to diminish assayable ACC activity is independent of nutritional state.