Venom isolated from Nasonia vitripennis was found to possess phenoloxidase (PO) activity. Enzyme activity was detected by using a modified dot blot analysis approach in which venom samples were applied to nylon membranes and incubated with either L-DOPA or dopamine. Dot formation was most intense with dopamine as the substrate and no activators were necessary to evoke melanization. No melanization occurred when venom was incubated in Schneider’s insect medium containing fetal bovine serum or tyrosine, but melanization did occur when larval or pupal plasma from the fly host, Sarcophaga bullata, was exposed to tyrosine. Only fly larval plasma induced an enzyme reaction with the Schneider’s insect medium. The PO inhibitors phenylthiourea (PKU) and phenylmethylsulfonylfluoride (PMSF) abolished PO activity in venom and host plasma samples, but glutathione (reduced) only inhibited venom PO. Elicitors of PO activity had no effect on the ability of venom, or larval and pupal plasma to trigger melanization reactions. SDS-PAGE separation of venom followed by in-gel staining using dopamine as a substrate revealed two venom proteins with PO activity with estimated molecular weights of 68 and 160 kilodaltons. In vitro assays were performed to determine the importance of venom PO in triggering cellular changes and evoking death. When cell monolayers were pre-treated with 10 mM PKU or PMSF prior to venom exposure, the cells were protected from the effects of venom as evidenced by no observable cellular morphological changes and over 90% cell viability by 24 h after venom treatment. Simultaneous addition of inhibitors with venom or lower concentrations of PMSF were less effective in affording protection. These observations argue that venom PO is unique from that of the fly hosts, and that the venom enzyme is critical to the intoxication pathway leading to cell death.