We identified 1-(5 chloronaphthalenesulfonyl)-1H-hexahydro-1,4-diazepine, also known as ML-9, as a powerful inhibitor of PKB activity in different cells as well as of recombinant PKB, It also inhibits other downstream serine/threonine kinases, such as PKA and p90 Se kinase, but not upstream tyrosine phosphorylation or PI3-kinase activation in response to insulin, We compared the effects of ML-9 and wortmannin on several insulin-stimulated effects in isolated rat fat cells. Both ML-9 and wortmannin inhibited glucose transport and GLUT4/IGF II receptor translocation to the plasma membrane. In contrast, only wortmannin inhibited the antilipolytic effect and PDE3B activation by insulin. Thus, ML-9 inhibits PKB but not PI3-kinase activation in response to insulin and is useful to differentiate between these effects, Both PI3-kinase and PKB are important for glucose transport and intracellular protein translocation while PKB does not appear to play an important role for the antilipolytic effect or activation of PDE3B in response to insulin.