O n the hypothesis that prostaglandins and other eicosanoids mediate nodulation responses to bacterial infections in insects, we describe an intracellular phospholipase A2 (PLA2) in homogenates prepared from hemocytes collected from the tobacco hornworm, Manduca sexta. PLAz hydrolyzes fatty acids from the sn-2 position of phospholipids. Some PlA2s are thought to be the first and rate-limiting step in biosynthesis of prostaglandins and other eicosanoids. The hemocyte PLA2 activity was sensitive to hemocyte homogenate protein concentration (up to 250 pg protein/reaction), pH (optimal activity at p H 8.0), and the presence of a Ca" chelator. Like PlA2s from mammalian sources, the hemocyte PLA2 was inhibited by the phospholipid analog oleyoxyethyl phosphorylcholine. Whereas most intracellular PLA2s require Ca2' for catalytic activity, some PLA2s, including the hemocyte enzyme, are Ca2'-independent. The hemocyte PLA2 exhibited a preference for arachidonyl-associated substrate over palmitoyl-associated substrate. These findings show that M. sexta hemocytes express a PLA2 that shows a marked preference for hydrolyzing arachidonic acid from phospholipids. The biological significance of this enzyme relates to cellular immune responses to bacterial infections. The hemocyte PIAP may be the first biochemical step in synthesis of the eicosanoids that mediate cellular itnmunity in insects.