Recent studies have suggested that abnormal regulation of protein phosphatase 2A (PP2A) is associated with type 2 diabetes in rodent and human tissues. Results with cultured mouse myotubes support a mechanism for palmitate activation of PP2A leading to activation of glycogen synthase kinase 3 (GSK3). Phosphorylation and inactivation of glycogen synthase by GSK3 could be the mechanism for long chain fatty acid inhibition of insulin-mediated carbohydrate storage in insulin resistant subjects. Here we test the effect of palmitic acid on cultured muscle glycogen synthase and PP2A activity. Palmitate inhibition of glycogen synthase fractional activity (GSFA) is increased in high body mass index (BMI) compared to lower BMI subjects (r=-0.43, p=0.03). Palmitate action on PP2A varies from inhibition in subjects with decreased 2 hour plasma glucose concentration (2HPG) to activation in subjects with increased 2HPG (r=0.45, p<0.03) during an oral glucose tolerance test. The results do not show an association between palmitate effects on PP2A and GSFA. We conclude that subjects at risk for type 2 diabetes have intrinsic differences in palmitate regulation of at least two enzymes (PP2A and glycogen synthase) that contribute to abnormal insulin regulation of glucose metabolism.