At least two signalling systems have the potential to contribute to the activation of protein kinase C (PKC) family members such as PKCepsilon. One of these is phosphoinositide 3-kinase (PI 3-kinase), whose lipid products activate PKCepsilon in vitro and in living cells. The recent observation that there are multiple waves of PI 3-kinase and PKCepsilon activity within the G(0)-to-S phase interval provides a new opportunity to investigate the relationship between these two signalling enzymes in vivo. We have assessed the relative importance of the early and late waves of PI 3-kinase activity for the corresponding waves of PKCepsilon activity. Blocking the first phase of PI 3-kinase activity inhibited both early and late activation of PKCepsilon. In contrast, the second wave of PI 3-kinase activity was dispensable for late activation of PKCepsilon. These findings suggested that early PI 3-kinase activation induced a stable change in PKCepsilon, which predisposed it to subsequent activation by lipid cofactors. Indeed, partial proteolysis of PKCepsilon indicated that early activation of PI 3-kinase led to a conformation change in PKCepsilon that persisted as the activity of PKCepsilon cycled. We propose a two-step hypothesis for the activation of PKCepsilon in vivo. One step is stable and depends on PI 3-kinase, whereas the other is transient and may depend on the availability of lipid cofactors. Finally, these studies reveal that PI 3-kinase and PKCepsilon are capable of communicating over a relatively long time interval and begin to elucidate the mechanism.