Phospholipase D (PLD) is known to play a role in vesicle transport through the hydrolysis of phosphatidyleholine (PC) to produce the bioactive lipid, phosphatidic acid. Lipid droplets (LDs) are surrounded by a monolayer of phospholipids, including PC and its lyso derivative, and exhibit a number of signaling proteins. Our recent report suggests that the association of adipose differentiation-related protein (ADRP) to LDs is regulated by an ADP-ribosylation factor 1 (Arf1)-dependent mechanism. In the present study, we found an increase in PLD activity accompanied with LD formation in oleic acid-treated NIH3T3 cells. Brefeldin A, an inhibitor of ARF-GEFs, suppressed both PLD activation and LD formation in oleic acid-treated cells. PLD1, but not PLD2, was found to exist in LDs by immunocytochemical analysis. Furthermore, co-existence of PLD1, Arf1, and ADRP was observed in the LD-enriched subcellular fractions obtained from oleic acid-treated NIH3T3 cells by Western blot analysis. PLD1 activity in the LD-enriched fractions was stimulated by exogenously added Arf1 Although LDs were induced in either PLD1- or PLD2-overexpressing CHO cells by oleic acid treatment, the stimulation of PLD activity was observed only in PLD1-CHO cells. Taken together, the data suggest that the activation of Arf1-dependent PLD1 occurs in LDs and may be involved in their physiological function. (c) 2005 Elsevier Inc. All rights reserved.