Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), inhibited adipocyte differentiation of 3T3-L1 fibroblasts induced by insulin/dexamethasone/IBMX (hormones/IBMX). Insulin as a key factor in the process of the adipocyte differentiation activated PI 3-kinase, Ras protein, and mitogen-activated protein kinase (MAP kinase, ERK) in 3T3-L1 fibroblasts. Pretreatment with wortmannin almost completely suppressed all these activations. These findings suggest that the sequential activation of PI 3-kinase, Ras protein, and MAP kinase is involved in the insulin signaling pathway(s) during differentiation by hormones/IBMX and in consequence of the inhibition of PI 3-kinase by wortmannin, the activation of Ras protein and MAP kinase which acts downstream of PI 3-kinase is suppressed and results in the inhibition of adipocyte differentiation.