Correlation between translocation and down‐regulation of conventional protein kinase Cα (cPKCα) and new PKCδ (nPKCδ) induced by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) at different time courses (5 min, 30 min, 1 h, 3 h, 6 h, 10 h, 17 h, and 24 h) was studied in C6 glioma cells. From the dose‐dependent translocations of these two isoforms by 10‐min treatment with TPA (1, 3, 10, 30, 100, 300, and 1,000 nM), we found that cPKCα was translocated by 3–1,000 nM and nPKCδ was translocated by 10–1,000 nM TPA. Both isoforms were maximally translocated by 100–1,000 nM TPA, whereas 1 nM did not translocate these two isoforms. When the cells were treated with 1,000 nM TPA for 5 min to 17 h, the translocation of these two isoforms occurred rapidly after 5‐min treatment and could be sustained for 1 h, whereas down‐regulation occurred after 3‐h treatment and almost complete down‐regulation was observed after 17‐h treatment. However, the extent of down‐regulation of nPKCδ was greater than that of cPKCα at 3‐, 6‐, and 10‐h treatment. Further studies by using different doses of TPA (100, 10, 3, and 1 nM) and extending the time to 24 h showed that cPKCα was more resistant to down‐regulation. This conventional isoform was maintained at a translocation state even after long‐term treatment with 3–100 nM TPA, and complete down‐regulation was only shown after 1,000 nM treatment. On the other hand, nPKCδ was almost completely down‐regulated by long‐term treatment with a translocation dose of 10–1,000 nM TPA despite higher membrane content of this new isoform. Therefore, the differential translocation and down‐regulation of cPKCα and nPKCδ was demonstrated in C6 glioma cells and this will be useful for exploring cPKCα‐ or nPKCδ‐specific functional roles in cellular functions and different signal transduction pathways in these cells.