Genetic approaches have shown that protein kinase B (PKB or Akt) is an important regulator of normal heart functions and dysregulation causes cardiac disease. Overexpression of constitutively activated PKB increases heart size and causes dilated myopathy. Insulin is a powerful activator of PKB whereas adrenaline is not considered a major physiological regulator of PKB in heart. However, we recently reported that adrenaline strongly potentiated insulin-stimulated PKB activation in skeletal muscles without having effect in the absence of insulin. The aim of the present study was to investigate the effect of isoprenaline on insulin-stimulated PKB phosphorylation in heart cells from adult rats. Isoprenaline increased insulin-stimulated PKB Ser⁴⁷³ and Thr³⁰⁸ phosphorylation > 3-fold in cardiomyocytes from adult rats. Isoprenaline alone did not increase PKB phosphorylation. Isoprenaline also increased insulin-stimulated GSK-3b Ser⁹ phosphorylation [asymp] 2-fold. PKB phosphorylates GSK-3b at Ser⁹; our data, therefore, support that isoprenaline increases insulin-stimulated PKB activity. Dobutamine (b₁-agonist) increased insulin-stimulated PKB phosphorylation as effectively as isoprenaline (> 3-fold) whereas salbutamol (b₂-agonist) only potentiated insulin-stimulated PKB phosphorylation by [asymp] 80 %. Dobutamine, but not salbutamol, increased phospholamban Ser¹⁶ phosphorylation and glycogen phosphorylase activation (PKA-mediated effects). Furthermore, the cAMP analogues that activate PKA (dibutyryl-cAMP and N⁶-benzoyl-cAMP) increased insulin-stimulated PKB phosphorylation by > 3-fold without effect alone. The Epac specific activator 8-(4-chlorophenylthio)-2`-O-methyl-cAMP (007) increased insulin-stimulated PKB phosphorylation by [asymp] 50 %. Db-cAMP and N⁶-benzoyl-cAMP, but not 007, increased phospholamban Ser¹⁶ phosphorylation. In conclusion, b-adrenoceptors are strong regulators of PKB phosphorylation via cAMP and PKA when insulin is present. We hypothesise that PKB mediates important signalling in the heart during b-adrenergic receptors stimulation.