Protein kinase C (PKC) is activated by 1,2-sn-diacylglycerol (DAG), the source of which can either be phosphatidylinositol bisphosphate or phosphatidylcholine. Here, we show that monogalactosyl diglyceride (MGDG), a minor galactolipid present in oligodendrocytes (OLs) and myelin, which is designated as a marker for myelination, can enhance OL PKC activity. Based on different calcium and substrate requirements we conclude that MGDG and DAG activate different isoforms of PKC group A: MGDG primarily stimulates PKC-alpha, and DAG primarily activates PKC-gamma. The presence of these PKC isoforms in OLs was confirmed by western blotting, whereas PKC-beta was only weakly stained, if at all. Addition of MGDG to the culture medium provided a higher density of regenerating OL fibers, which was not observed when membrane-permeable DAG was used. These findings indicate that MGDG can modulate the OL PKC activity and that PKC-alpha is the major PKC isoform involved in OL process formation.