Phospholipase B (PLB) activity was found in a variety of snake, Gila monster and Hymenoptera venoms. Hymenoptera venoms, except bee, are an especially rich source of PLB. This was shown by incubation of crude venoms with lysophosphatidylcholine and subsequent titration of liberated fatty acids. PLB activity was not found in pure α- or β-bungarotoxin, α-cobrotoxin, or one crude snake venom ( Crotalus horridus horridus). Snake, Gila monster and bee venoms exhibit higher PLB activity at an alkaline pH, while hornet venom PLB activity is greater at a neutral pH. Phospholipase A 2 (PLA 2) activity was determined using phosphatidylcholine and egg yolk as substrates. In reptile and bee venoms PLA 2 activity is much higher than PLB activity. In contrast, hornet venom PLA 2 and PLB activities are nearly equal. In reptile venoms PLA 2 activity is optimal at neutral pH, in contrast to PLB activity, suggesting that separate proteins or active sites are responsible for PLA 2 and PLB activities. The effect of boiling or heating venoms in either an acidic or alkaline milieu upon subsequent PLA 2 and PLB activity, measured at 37°C, was examined. The PLA 2 and PLB activities of all venoms tested were lost upon boiling at pH 9.4, except for the PLA 2 activity of Notechis scutatus venom which retained about 30% of its activity. Boiling at pH 5.5 results in greatly varying extents of retention of PLA 2 and PLB activities, dependent upon the venom examined. Therefore, the heat stability characteristics for each venom must be experimentally determined, not assumed. Boiling destroys PLA 2 and PLB activities of oriental hornet venom at about the same rate. No conclusive results were obtained from the heating studies as to whether PLA 2 and PLB activities reside upon the same molecule. However, PLA 2 and PLB activities of oriental hornet venom were separated using triple tandem column gel permeation chromatography, demonstrating the existence of two separate proteins for these activities.