In this work, we examined the neuromuscular activity of Bothriopsis bilineata smargadina (forest viper) venom in vertebrate isolated nerve-muscle preparations. In chick biventer cervicis preparations the venom caused concentration-dependent (0.1-30μg/ml) neuromuscular blockade that was not reversed by washing, with 50% blockade occurring in 15-90min. Muscle contractures to exogenous acetylcholine and KCl were unaffected by venom, but there was a slight increase in creatine kinase release after 120min (from 80±15 to 206±25U/ml, n=6, p<0.05). In mouse phrenic nerve-diaphragm preparations, the venom (1, 10 and 30μg/ml) produced marked facilitation (∼120% increase above basal) at the highest concentration followed by neuromuscular blockade; the effects at lower concentrations were considerably less marked. Venom increased the quantal content values after 15 and 30min followed by significant inhibition at ≥90min. However, venom did not alter the muscle membrane resting potential or the response to exogenous carbachol. In both preparations, incubation at 22°C instead of 37°C delayed the onset of blockade, as did inhibition of venom PLA 2 activity. In curarized mouse preparations, the venom produced only muscle facilitation. These results indicate that B. b. smargadina venom causes neuromuscular blockade in vitro by a presynaptic mechanism involving PLA 2. © 2011 Elsevier Ltd.