Unsaturated fatty acids, such as arachidonic acid, have been implicated as second messengers of various cellular responses. In this paper, we demonstrate that the release of arachidonate from human polymorphonuclear leukocytes (PMNL) via a pathway initiated by phospholipase D (PLD) mediates phorbol myristate acetate (PMA)-induced desensitization of leukotriene B4 (LTB4) receptors. PMA caused a delayed release of arachidonic acid from PMNL, which occurred within minutes of the generation of the PLD products, phosphatidic acid and diglyceride, from [3H]lysophosphatidylcholine prelabeled PMNL. Moreover, stimulating PMNL with PMA in the presence of ethanol resulted in the formation of phosphatidylethanol at the expenses of phosphatidic acid, diglyceride and arachidonic acid. The PMA-induced generation of these three PLD products was inhibited by mepacrine which, in parallel, significantly blocked PMA-induced desensitization of [3H]LTB4 binding, which suggested that elevation of one or more of these products played a role in desensitization. In contrast, under basal conditions mepacrine reduced levels of these three lipids in a dose-related manner and, in parallel, increased basal [3H]LTB4 binding to PMNL. The reduction of PMA-induced LTB4 receptor desensitization by mepacrine could be overcome to various degrees by adding back PLD-derived lipids such as arachidonic acid. These data demonstrate that [3H]LTB4 binding to PMNL is decreased by PLD-derived lipids and suggests that intracellular arachidonic acid may modulate PMNL responsiveness to LTB4 activation.