The importance of E-cadherin protein in the establishment and maintenance of homotypic contacts in epithelial cells has already been determined. We report here that the association of E-cadherin to cytoskeleton, required for the functionality of this protein, increases progressively after seeding; in HT-29 M6 cells 4–5 days were required for detecting most of E-cadherin in the Triton X-100-insoluble (cytoskeleton-associated) fraction. The phorbol ester TPA differently affected E-cadherin levels in HT-29 M6 cells; at day 2–3, when most E-cadherin was found not-associated to the cytoskeleton, very important decreases (90%) in the total levels of this protein were detected as soon as 6 h after the addition of this compound. However, on later days (day 5), the predominant effect by 6 h was a translocation from the Triton-insoluble to the soluble fraction. The E-cadherin-associated proteins α-catenin and β-catenin were not significantly affected by treatment with TPA.