Insulin inhibits apolipoprotein B (apoB) secretion by primary rat hepatocytes through activation of phosphoinositide 3-kinase (PI 3-K). Current studies demonstrate that the PI 3-K inhibitor wortmannin inhibits both basal and insulin-stimulated PI 3-K activities. Wortmannin and LY 294002, two structurally distinct PI 3-K inhibitors, prevent insulin-dependent inhibition of apoB secretion in a dose-dependent manner. To link PI 3-K activation to insulin action on apoB, we investigated whether insulin induced localization of activated PI 3-K to the endoplasmic reticulum (ER), where apoB biogenesis is initiated. Insulin action results in a significant redistribution of PI 3-K to a low density microsome (LDM) fraction containing apoB protein and apoB mRNA. Insulin stimulates a significant increase in PI 3-K activity associated with insulin receptor substrate-1 as well as an increase in insulin receptor substrate-1/PI 3-K mass in LDM. Subfractionation of LDM on sucrose density gradients shows that insulin significantly increases the amount of PI 3-K present in an ER fraction containing apoB. Insulin stimulates PI 3-K activity in smooth and rough microsomes isolated from rat hepatocytes, the latter of which contain rough ER as demonstrated by electron microscopy. Studies indicate that 1) PI 3-K activity is necessary for insulin-dependent inhibition of apoB secretion by rat hepatocytes; 2) insulin action leads to the activation and localization of PI 3-K in an ER fraction containing apoB; and 3) insulin stimulates PI 3-K activity in the rough ER.