The phosphoinositide 3-kinase (PI 3-kinase) signaling pathway has been shown to play a pivotal role in intracellular signal transduction pathways involved in cell growth, cellular transformation and tumorigenesis. Analysis of several colon adenocarcinoma cell lines indicates that the PI 3-kinase signaling pathway is up-regulated in colon cancers. In particular, the protein levels and phosphorylation status of Akt and p70 S6 kinase are up-regulated in colon adenocarcinoma cell lines. More significantly, we have demonstrated for the first time that the phosphorylation of FKHR, a downstream target of Akt, is increased in these cell lines. Intriguingly, phosphorylation of three components of the PI 3-kinase signaling pathway, namely Akt, p70 S6 kinase and FKHR, are in direct correlation with the degree of tumorigenic potential of the colon cell lines tested. No differences in the protein levels of the two subunits of PI 3-kinase, p85 and p110alpha, and PTEN were noted. Real-time quantitative PCR indicated an increase in levels of Akt message only, and not of the other signaling pathway components. Inhibition of the PI 3-kinase with wortmannin decreased the anchorage-independent growth of colon cells in a soft agar assay. Hence, the components of the PI 3-kinase signaling pathway could serve as potential candidates for drug development in treatment of colon cancer.