Accumulation of lipids in non-adipose tissues, particularly liver and skeletal muscle, is associated with the development of insulin resistance. However, it is not entirely clear whether ectopic lipid accumulation plays a causal role in the development of insulin resistance or whether this is simply an associative relationship. We have conducted a number of studies to explore this relationship. Firstly, to further understand the role of muscle lipids in mediating insulin action, we generated a muscle-specific knockout of a key enzyme in phospholipid synthesis, CTP:phosphoethanolamine cytidylyltransferase (ECT), which resulted in marked (2-3-fold) increases in both diacylglycerol and triacylglycerol content in muscle. Despite this increase in lipid content, whole body and skeletal muscle insulin sensitivity, as determined by euglycemic hyperinsulinemic clamp, was not altered. These findings demonstrate that lipid accumulation in muscle is not always associated with insulin resistance. To examine the role of hepatic lipids, we performed a study where chronically (8 wk) high-fat, high-sucrose fed (HFSD) mice were switched back to a standard chow diet for 7 days. Upon the switch, energy intake was reduced, resulting in reductions of fat mass and hepatic diacylglycerol and triacylglycerol content. However, these parameters were still elevated compared to chow-fed mice, thus representing an intermediate phenotype. Nonetheless, glucose intolerance and hyperinsulinemia were completely normalized in mice that underwent the 7 day diet switch. This indicates that lipotoxicity per se does not necessarily maintain the glucose intolerant and insulin resistant state in HFSD fed mice. Rather, it appears that persistent over nourishment is likely to be the major factor responsible for causing defects in glucose metabolism. Together, these findings dissociate tissue lipid accumulation from the development of insulin resistance and glucose intolerance.