Primary cultures of rat cortical astrocytes express phospholipase D (PLD) isoforms 1 and 2 as determined by RT-PCR and Western blot. Basal PLD activity was strongly (10-fold) increased by 4beta-phorbol-12beta,13alpha-dibutyrate (PDB) (EC(50): 56 nM), an effect which was inhibited by Ro 31-8220 (0.1-1 microM), an inhibitor of protein kinase C (PKC), and by brefeldin A (10-100 microg/ml), an inhibitor of ADP-ribosylating factor (ARF) activation. Pretreatment of the cultures with Clostridium difficile toxin B-10463 (0.1-1 ng/ml), which inactivates small G proteins of the Rho family, led to a breakdown of the astroglial cytoskeleton; concomitantly, PLD activation by PDB was reduced by up to 50%. In contrast, inactivation of proteins of the Ras family by Clostridium sordellii lethal toxin 1522 did not affect PLD activation. In parallel experiments, serum-induced PLD activation was sensitive to brefeldin A, but not to Ro 31-8220 and not to clostridial toxins. We conclude that, in astrocytes, the PLD isoform which is activated by phorbol ester requires PKC, ARF and Rho proteins for full activity and probably represents PLD1.