Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) synthesis has been implicated in maintaining the function of the Golgi apparatus. Here we demonstrate that the inhibition of PtdIns(4,5)P(2) synthesis in vitro in response to primary alcohol treatment and the kinetics of Golgi fragmentation in vivo were very rapid and tightly coupled. Preloading Golgi membranes with short chain phosphatidic acid abrogated the alcohol-mediated inhibition of PtdIns(4,5)P(2) synthesis in vitro. We also show that fragmentation of the Golgi apparatus in response to diminished PtdIns(4,5)P(2) synthesis correlated with both the phosphorylation of a Golgi form of beta III spectrin, a PtdIns(4,5)P(2)-interacting protein, and changes in its intracellular redistribution. The data are consistent with a model suggesting that the decreased PtdIns(4,5)P(2) synthesis and the phosphorylation state of beta III spectrin modulate the structural integrity of the Golgi apparatus.