Background: Prostate carcinoma has become the second most fatal cancer in American men. In Dunning R3327 rat prostate adenocarcinoma cells, elevated invasiveness positively correlates with metastatic potential. However, the mechanism(s) responsible for regulation of tumor cell motility and invasion is poorly understood. We have reported that a lipoxygenase metabolite of arachidonic acid 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], augments tumor cell metastatic potential through activation of protein kinase C (PKC). Purpose: We proposed to determine the effect of 12(S)-HETE on the motility and invasion of low-metastatic rat prostate AT2.1 tumor cells and the effect of 12(S)-HETE activation of specific PKC isform(s) in these processes. Methods: The motility of AT2.1 cells was determined by the colloidal gold phagokinetic track assay and the invasiveness measured as their ability to invade through basement membrane Matrigel-coated filters. Expression of PKC isoforms was determined by Western blotting of the whole cell lysate with isoformspecific anti-PKC antibodies. Cytosol and membrane fractions were prepared and the subcellular distribution of PKC was analyzed by Western blotting and activity assay. The effect of 12(S)-HETE on cell proliferation was examined. Data were analyzed for significance of difference with the two-sampled, two-sided Student's t test. Results: 12(S)-HETE increased the motility and invasion of AT2.1 cells, and this 12(S)-HETE—increased motilityand invasion were inhibited by a selective PKC inhibitor, calphostin C, as well as a Ca² chelator, bis-(o-aminophenoxy)ethane-N, N, N′, N′-tetraacetic acid/tetra (acetoxy-methyl)ester. AT2.1 cells expressed the PKC isoforms α and δ, and 12(S)-HETE increased the membrane association of PKCα but not δ. Further, the motility and invasion of AT2.1 cells were increased by thymelea toxin, a selective activator of PKCα over PKCδ. Conclusion: 12(S)-HETE augments the invasiveness of AT2.1 cells via selective activation of PKCα. Implications: 12(S)-HETEmodulation of PKCα invasiveness may be an important mechanism of action for the regulation of the invasive potential of rat prostate carcinoma cells, and the 12-lipoxygenase enzyme and/or PKCα may serve as key targets for the development of anti-invasive agents useful for combating the spread of prostate cancer.