Activated Akt/protein kinase B transmits oncogenic signals leading to inhibition of apoptosis, cellular proliferation, and tolerance to hypoxia. Presently, mutational inactivation of PTEN and activation of Ras are considered to be the major causes of Akt activation. Here we report differential mechanisms of constitutive Akt activation in 4 human pancreatic cancer cell lines (KMP-3, KMP-4, PCI-66, and PCI-68). These 4 cell lines displayed phosphorylation and functional activation of Akt both in the presence and absence of serum, while three control cell lines (PCI-79, KMP-8, and PSN-1) did so only in the presence of serum in culture. All the 7 cell lines harbored K-Ras activated by mutations at codon 12 resulting in MAP kinase kinase (MEK1/2) phosphorylation, and all except one (KMP-8) had p53 mutations, indicating that these mutations are not sufficient for constitutive Akt activation. KMP-3 and KMP-4 had lost PTEN function owing to loss of expression or a mutation, but PCI-66 and PCI-68 retained wild-type PTEN. Phosphorylation of Akt was inhibited by the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 and the tyrosine kinase inhibitor genistein in KMP-3 and KMP-4 cells, indicating that upstream signals are required for Akt activation in these two cell lines. In contrast, neither LY294002 nor genistein inhibited Akt activation in PCI-66 and PCI-68 cells, indicating the involvement of another unknown mechanism of Akt activation independent of PI3K-mediated signaling to Akt. Irrespective of the differential mechanisms, the 4 cell lines showed similar mRNA expression patterns of 49 genes assessed by cDNA array as compared to the 3 cell lines without Akt activation, suggesting that the mechanisms have the same consequences on the downstream signaling of the constitutive Akt activation.