The neuromuscular activity of Bbil-TX, a PLA2 with catalytic activity isolated from Bothriopsis bilineata smargadina venom, was examined in chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations. In BC preparations, Bbil-TX (0.5-10μg/ml) caused time- and concentration-dependent blockade that was not reversed by washing; the times for 50% blockade were 87±7, 41±7 and 19±2min (mean±SEM; n=4-6) for 1, 5 and 10μg/ml, respectively. Muscle contractures to exogenous ACh and KCl were unaffected. The toxin (10μg/ml) also did not affect the twitch-tension of directly-stimulated, curarized (10μg/ml) BC preparations. However, Bbil-TX (10μg/ml) produced mild morphological alterations (edematous and/or hyperchromic fibers) in BC; there was also a progressive release of CK (from 116±17IU/ml (basal) to 710±91IU/ml after 45min). Bbil-TX (5μg/ml)-induced blockade was markedly inhibited at 22-24°C and pretreatment with p-bromophenacyl bromide (p-BPB) abolished the neuromuscular blockade. Bbil-TX (3-30μg/ml, n=4-6) caused partial time- and concentration-dependent blockade in PND preparations (52±2% at the highest concentration). Bbil-TX (30μg/ml) also markedly reduced the MEPPs frequency [from 26±2.5 (basal) to 10±1 after 60min; n=5; p<0.05] and the quantal content [from 94±14 (basal) to 24±3 after 60min; n=5; p<0.05] of PND preparations, but caused only minor depolarization of the membrane resting potential [from -80±1mV (basal) to -66±2mV after 120min; n=5; p<0.05], with no significant change in the depolarizing response to exogenous carbachol. These results show that Bbil-TX is a presynaptic PLA2 that contributes to the neuromuscular blockade caused by B. b. smargadina venom. copy; 2013 Elsevier Ltd.