In this study isolated perfused working rat hearts were used to investigate the role of palmitate-regulated protein kinase B (PKB) phosphorylation on glucose metabolism. Rat hearts were perfused aerobically in working mode with 11 mM glucose and either 100 μU/ml insulin or 100 μU/ml insulin and 1.2 mM palmitate. PKB activity and phosphorylation state were reduced in the presence of 1.2 mM palmitate, which correlates with a decrease in glycolysis (47%), glucose oxidation (84%), and glucose uptake (43%). In contrast to skeletal muscle, neither p38 nor ERK underwent changes in their phosphorylation states in response to insulin or insulin and palmitate. Moreover, pharmacological restoration of glucose oxidation rates in hearts perfused with 1.2 mM palmitate demonstrated no increase in PKB phosphorylation state. In cultured mouse cardiac muscle HL-1 cells, insulin markedly increased PKB phosphorylation, which was blunted by pre- and cotreatment with 1.2 mM palmitate. However, neither palmitate nor C₂-ceramide treatment of insulin-stimulated cells was able to accelerate PKB dephosphorylation beyond that observed following the removal of insulin alone. Taken together, these experiments show the control of PKB phosphorylation by palmitate is independent of ceramide and suggest that this signaling event may be an important regulator of myocardial glucose uptake and oxidation.