The role of low levels of phospholipase A 2 (PLA 2) activity and intracellular Ca 2+ stores in the pharmacological action of bothropstoxin (BthTX), a myotoxic Lys49 PLA 2 homologue isolated from the venom of Bothrops jararacussu, was investigated. We examined the muscular effects of BthTX in the mouse diaphragm and its PLA 2 activity in radiolabeled human and rat primary cultures of skeletal muscle. Although it is a Lys49 PLA 2 homologue, BthTX had a low, but easily detectable, level of enzymatic activity relative to two Asp49 PLA 2 enzymes from Naja naja kaouthia and Naja naja atra venoms, and this activity was reduced by about 85% in the presence of Sr 2+ (4.0 mM). However, the replacement of 1.8 mM Ca 2+ by 4 mM Sr 2+ did not alter the BthTX-induced contracture and blockade of the muscle twitch tension. In addition, Sr 2+ decreased by 50% the time required to cause 50% paralysis, and evoked approximately a four-fold increase in the number of spontaneous spikes. In isolated sarcoplasmic reticulum preparations, BthTX opened the intracellular Ca 2+ release channel (ryanodine receptor) and lowered the threshold of Ca 2+-induced Ca 2+ release by a second, as yet unidentified, mechanism. However, in intact muscle, dantrolene, an antagonist of some forms of intracellular Ca 2+ release, had no effect on the actions of BthTX. These findings do not support any role for the low levels of PLA 2 activity, or dantrolene-sensitive intracellular Ca 2+ stores, in the action of BthTX. The mechanism whereby Sr 2+ stimulates the pharmacological activity of BthTX remains to be clarified.