The protein kinase C (PKC) is a family of serine/threonine kinases that are key regulatory enzymes involved in growth, differentiation, cytoskeletal reorganization, tumor promotion, and migration. We investigated the functional involvement of PKC isotypes and of E-cadherin in the regulation of the locomotion of six human colon-adenocarcinoma cell lines. The different levels of the PKC α and the E-cadherin expression have predictable implications in the spontaneous locomotory activity. With the use of PKC α–specific inhibitors (safingol, Go6976) as well as the PKC δ–specific inhibitor rottlerin, we showed that only PKC α plays a major role in the regulation of tumor cell migration. The results were verified by knocking out the translation of PKC isozymes with the use of an antisense oligonucleotide strategy. After stimulation with phorbol ester we observed a translocation and a colocalization of the activated PKC α at the plasma membrane to the surrounding extracellular matrix. Furthermore, we investigated the functional involvement of E-cadherin in the locomotion with the use of a blocking antibody. A high level of PKC α expression together with a low E-cadherin expression was strongly related to a high migratory activity of the colon carcinoma cells. This correlation was independent of the differentiation grade of the tumor cell lines.