Arachidonic acid (AA) release via phospholipase A2 (PLA2) activation and generation of eicosanoids have been implicated as playing signaling roles in a variety of cell types. Here we show evidence that interaction of fresh NK cells with membranes from sensitive or antibody (Ab)-coated targets generates eicosanoids through both cyclooxygenase (CO) and lipoxygenase (LO) pathways. Eicosanoid generation is attributable to PLA2 activation since pretreatment with PLA2 irreversible inhibitors, such as mepacrine or para-bromophenacylbromide (pBPB), completely blocks AA metabolite generation. The involvement of PLA2 or AA metabolites in the cytotoxic functions of rat NK cells has also been investigated. Treatment of effector cells with mepacrine or pBPB resulted in complete, irreversible, dose-dependent inhibition of both NK and ADCC activities, which were completely reversed by the addition of exogenous PLA2 or its hydrolysis products, AA and lysophosphatidylcholine (lysoPC). Among the metabolites of AA released by NK cells, the 5-LO product leukotriene B4 (LTB4) seems to play an important role in cytolytic activities of NK cells. Indeed, the LO inhibitor, nordihydroguaiaretic acid (NDGA), totally abrogated both NK and ADCC activities, which were restored by the addition of exogenous LTB4. However, the failure of LTB4 to reverse mepacrine or pBPB-induced inhibition of NK and ADCC suggests that its effects could be mediated by PLA2. The results are consistent with a crucial role for the target-stimulated AA release as a fundamental step in the signal transduction pathway in NK cell. Moreover, LTB4 generation seems to be responsible for further PLA2 activation in a second step leading to the amplification of response.