The present study focuses on a putative regulation of PKCbetaII by phosphatidylinositol-3 kinase (PI 3-kinase) in colorectal carcinoma cells; little is known about the role and activity of PKCbetaII in these cells. We examined the activity of PI 3-kinase in two adenocarcinoma cell lines, HT29 cells that differentiate only after stimulation with appropriate agents, and Caco-2 cells that can differentiate spontaneously. The activity of PI 3-kinase as well as the activity of PKCbetaII appeared to decrease only in HT29 cells in which differentiation was induced by sodium butyrate. In HT29 cells infected with recombinant adenovirus encoding constitutively active PI 3-kinase, the activity of alkaline phosphatase was almost completely blocked, and this PI 3-kinase significantly potentiated the activity of PKCbetaII in HT29 cells despite the presence of NaBT in the culture medium. On the contrary, in differentiating Caco-2 cells, the activity of PI 3-kinase was not butyrate-sensitive. In agreement with these findings, the alkaline phosphatase activity was not affected by constitutively active PI 3-kinase overexpressed in Caco-2 cells. These observations suggest that PKCbetaII is regulated by PI 3-kinase in HT29 cells and that the mechanisms of spontaneous differentiation versus butyrate-induced differentiation of adenocarcinoma cells may be different.