It was recently demonstrated that an increase in the cellular cholesterol level leads to vesiculation of the Golgi apparatus. This vesiculation affects the entire Golgi apparatus and is a reversible process. We have now started to elucidate the mechanism behind this cholesterol-induced vesiculation of the Golgi apparatus. Transient transfection of cells with dominant negative mutant constructs of dynamin 1 and 2 inhibited the vesiculation; expression of dynK44A in HeLa cells stably transfected with this construct had the same effect. However, the vesiculation seems to be independent of clathrin, as cholesterol-induced vesiculation still occurred following knock down of clathrin heavy chain in HeLa cells using RNA interference as well as in BHK cells where expression of antisense to clathrin heavy chain had been induced. Importantly, the cPLA2 inhibitor MAFP and the chelator BAPTA-AM that binds cytosolic Ca2+ inhibited the cholesterol-induced vesiculation, suggesting involvement of a cPLA2 that requires cytosolic Ca2+ for translocation to membranes. Furthermore, in response to an increased cellular cholesterol level, an EGFP-cPLA2 fusion protein translocated to the Golgi apparatus. Thus, our results demonstrate that the cholesterol-induced vesiculation of the Golgi apparatus is mediated by a cPLA2- and dynamin-dependent mechanism.