Rat Leydig cells contain a phospholipase D (PLD), which can be activated by vasopressin and phorbol ester. In order to clarify which Leydig cell organelles that express PLD activity, the subcellular localization of two differently regulated PLD activities was investigated by subcellular fractionation on a 40% (v/v) self-generating Percoll gradient. PLD activities in broken cells were estimated using radiolabeled didecanoylphosphatidylcholine as a substrate. Initial experiments revealed the presence of an oleate Mg²⁺-activated PLD and a phosphatidylinositol 4,5-bisphosphate-activated PLD (PIP₂-PLD) in the microsomal fraction of Leydig cells. The latter activity could be further stimulated by recombinant nonmyristoylated ADP ribosylating factor 1 (ARF1) plus GTPγS. The peak of oleate Mg²⁺ - PLD activity colocalized with the plasma membrane marker, whereas the highest specific activity of the PIP₂-PLD activity was found in fractions with a slightly lower density than those containing the plasma membrane and trans-Golgi marker enzymes. In order to localize phorbol ester- stimulated PLD activity in intact Leydig cells, the cells were prelabeled with [¹⁴C]-palmitate and then stimulated for 15 min with 100 nM 4-β- phorbol-12-myristate-13-acetate (PMA) in the presence of ethanol or butanol. The PLD product [¹⁴C]-phosphatidylethanol, expressed as the percentage of total labeled phospholipids in the fraction, was slightly increased in all Percoll fractions and showed a prominent peak in the fractions containing plasma membrane, trans-Golgi, and fractions of slightly lower density. The PMA-induced formation of [¹⁴C]-phosphatidylbutanol could be inhibited dose- dependently with brefeldin A suggesting that the activation of PLD by the phorbol ester was mediated by ARF.