Metastasis requires cytoskeletal remodeling for migration, adhesion, and extravasation of metastatic cells. Although protein kinase C (PKC) is involved in tumor promotion/progression and cytoskeletal remodeling, its role in metastasis has not been defined. PKCδ levels are increased in highly metastatic 13762NF mammary tumor cells (MTLn3) compared with less metastatic, parental cell lines. To determine whether the increase in endogenous PKCδ is functionally related to their increased metastatic potential, we prepared MTLn3 cells that express the inhibitory regulatory domain fragment of PKCδ (RDδ) under the control of a tetracycline-inducible promoter. RDδ expression attenuated endogenous PKC activity, as demonstrated by decreased phosphorylation of the PKC substrate adducin in migrating cells. Thus, in MT cells, RDδ appears to primarily influence cytoskeleton-dependent processes rather than cell cycle progression. To determine whether RDδ expression influenced metastatic potential in vivo, MTLn3/RDδ cells were either grown in the mammary fat pad or injected into the tail vein of syngeneic rats, and effects of doxycycline-induced RDδ expression on pulmonary metastases were studied. Consistent with the in vitro data, induction of RDδ significantly reduced the number of lung metastases without affecting growth of the primary tumor. These results suggest that interfering with endogenous PKCδ activity by expressing the inhibitory RDδ fragment inhibits cytoskeleton-regulated processes important for MTLn3 cell metastasis.