The present study investigates the roles of the spinal phosphoinositide 3-kinase (PI3K) signaling pathway in naloxone-precipitated withdrawal in acute and chronic morphine-dependent mice. There are two principal findings: (1) intrathecal pretreatment with wortmannin or LY294002, two structurally unrelated PI3K inhibitors, produced a dose-dependent increase of naloxone-precipitated withdrawal jumping, which was accompanied by an increased expression of spinal Fos protein in acute and chronic morphine-dependent mice; and (2) the expression of spinal p110gamma, the catalytic subunit PI3K, in the membrane fraction was significantly down-regulated by naloxone-precipitated withdrawal in acute and chronic morphine-dependent mice. This study provides new evidence showing that inactivation of the PI3K signaling pathway in the spinal cord may be involved in the expression of morphine withdrawal.