T he Golgi complex and the trans-Golgi network are critical cellular organelles involved in the endocytic and biosynthetic pathways of protein trafficking. Lipids have been implicated in the regulation of membrane-protein trafficking, vesicular fusion, and targeting. We have explored the role of cytosolic group IV phospholipase A₂ (cPLA₂) in membrane-protein trafficking in kidney epithelial cells. Adenoviral expression of cPLA₂ in LLC-PK₁ kidney epithelial cells prevents constitutive trafficking to the plasma membrane of an aquaporin 2-green fluorescent protein chimera, with retention of the protein in the rough endoplasmic reticulum. Plasma membrane Na⁺-K⁺-ATPase α-subunit localization is markedly reduced in cells expressing cPLA₂, whereas the trafficking of a Cl^–/HCO₃^– anion exchanger to the plasma membrane is not altered in these cells. Expression of cPLA₂ results in dispersion of giantin and β-COP from their normal, condensed Golgi localization, and in marked disruption of the Golgi cisternae. cPLA₂ is present in Golgi fractions from noninfected LLC-PK₁ cells and rat kidney cortex. The distribution of tubulin and actin was not altered by cPLA₂, indicating that the microtubule and actin cytoskeleton remain intact. Total cellular protein synthesis is unaffected by the increase in cPLA₂ activity. Thus cPLA₂ plays an important role in determining Golgi architecture and selective control of constitutive membrane-protein trafficking in renal epithelial cells.