Factors that stimulate mitochondrial biogenesis in skeletal muscle include AMPK, calcium, and circulating FFAs. Chronic treatment with either AICAR, a chemical activator of AMPK, or increasing circulating FFAs with a high fat diet increases mitochondria in rat skeletal muscle. The purpose of this study was to determine whether the combination of chronic chemical activation of AMPK and high fat feeding would have an additive effect on skeletal muscle mitochondria levels. We treated Wistar male rats with a high fat diet (HF), AICAR injections (AICAR), or a high fat diet and AICAR injections (HF+AICAR) for six weeks. At the end of the treatment period, markers of mitochondrial content were examined in white quadriceps, red quadriceps, and soleus muscles, predominantly composed of unique muscle-fiber types. In white quadriceps, there was a cumulative effect of treatments on LCAD, cytochrome c, and PGC-α protein, as well as on citrate synthase and β-HAD activity. In contrast, no additive effect was noted in the soleus and in the red quadriceps only β-HAD activity increased additively. The additive increase of mitochondrial markers observed in the white quadriceps may be explained by a combined effect of two separate mechanisms: high fat diet-induced post transcriptional increase in PGC-α protein and AMPK mediated increase in PGC-α protein via a transcriptional mechanism. These data show that chronic chemical activation of AMPK and a high fat diet have a muscle type specific additive effect on markers of fatty acid oxidation, the citric acid cycle, the electron transport chain, and transcriptional regulation.