Crotalus ruber ruber venom contains several different proteases, and the proteolytic activity of the crude venom is 6-15 times greater in adult than in juvenile venom. Venom samples were assayed for proteolytic, phosphodiesterase, L-amino acid oxidase and elastinase-like activities and were subjected to gel filtration on BioGel P-100. Two major size classes of proteases were resolved (mol. wt 67,000 and 20,500). EDTA, N-ethylmaleimide (N-EM) and 1,10-phenanthroline inhibited proteolytic activity of crude venom, and EDTA, Zn2+ and Cu2+ inhibited proteolytic activity of the fractionated venom.