Characterization of venom (Duvernoy's secretion) from twelve species of colubrid snakes and partial sequence of four venom proteins. Toxicon XX, xx±yy, 2000. Ð Venomous colubrids, which include more than 700 snake species worldwide, represent a vast potential source of novel biological compounds. The present study characterized venom (Duvernoy's gland secretion) collected from twelve species of opisthoglyphous (rear-fanged) colubrid snakes, an extremely diverse assemblage of non-venomous to highly venomous snakes. Most venoms displayed proteolytic activity (casein), though activity levels varied considerably. Low phosphodiesterase activity was detected in several venoms (Amphiesma stolata, Diadophis punctatus, Heterodon nasicus kennerlyi, H. n. nasicus and Thamnophis elegans vagrans ), and acetylcholinesterase was found in Boiga irregularis saliva and venom, but no venoms displayed hyaluronidase, thrombin-like or kallikrein-like activities. High phospholipase A2 (PLA2) activity was found in Trimorphodon biscutatus lambda venom, and moderate levels were detected in Boiga dendrophila and D. p. regalis venoms as well as B. dendrophila and H. n. nasicus salivas. Non-reducing SDS± PAGE revealed 7±20 protein bands (3.5 to over 200 kD, depending on species) for all venoms analyzed, and electrophoretic pro®les of venoms were typically quite distinct from saliva pro®les. Components from A. stolata, Hydrodynastes gigas, Tantilla nigriceps and T. e. vagrans venoms showed protease activity when run on gelatin zymogram gels. N-termina protein sequences for three 26 kD venom components of three species (H. gigas, H. torquata, T. biscutatus ) and one 3.5 kD component (T. nigriceps ) were also obtained, and the 3.5 kD peptide showed apparent sequence homology with human vascular endothelial growth factor; these data represent the ®rst sequences of colubrid venom components. Protease, phosphodiesterase and PLA2 activities are also common to elapid and viperid snake venoms, but it is apparent that numerous other (as yet undescribed) components make up the majority of colubrid venom proteins. The complex nature of venoms produced by most species surveyed, and the high levels of protease or phospholipase A2 activity of some venoms, suggest that many colubrids could become an important source of human health concern as encounters with these snakes increase.