We have previously isolated a Lys49 phospholipase A2 homolog (BaTX) from Bothrops alternatus snake venom using a combination of molecular exclusion chromatography and reverse phase HPLC and shown its ability to cause neuromuscular blockade. In this work, we describe a one-step procedure for the purification of this toxin and provide further details of its neuromuscular activity. The toxin was purified by reverse phase HPLC and its purity and molecular mass were confirmed by SDS-PAGE, MALDI-TOF mass spectrometry, amino acid analysis and N-terminal sequencing. BaTX (0.007-1.4 μM) produced time-dependent, irreversible neuromuscular blockade in isolated mouse phrenic nerve-diaphragm and chick biventer cervicis preparations (time to 50% blockade with 0.35 μM toxin: 58 ± 4 and 24 ± 1 min, respectively; n = 3-8; mean ± S.E.) without significantly affecting the response to direct muscle stimulation. In chick preparations, contractures to exogenous acetylcholine (55 and 110 μM) or KCl (13.4 mM) were unaltered after complete blockade by all toxin concentrations. These results, which strongly suggested a presynaptic mechanism of action for this toxin, were reinforced by (1) the inability of BaTX to interfere with the carbachol-induced depolarization of the resting membrane, (2) a significant decrease in the frequency and amplitude of miniature end-plate potentials, and (3) a significant reduction (59 ± 4%, n = 12) in the quantal content of the end-plate potentials after a 60 min incubation with the toxin (1.4 μM). In addition, a decrease in the organ bath temperature from 37 °C to 24 °C and/or the replacement of calcium with strontium prevented the neuromuscular blockade, indicating a temperature-dependent effect possibly mediated by enzymatic activity. © 2009 Elsevier Inc. All rights reserved.