The sting of the black scorpion Heterometrus spinifer, which can cause intense localized pain, has not been reported to produce lethal cardiovascular complications, which are well known to result from scorpion envenomation as a consequence of a massive release of catecholamines. Therefore, we have undertaken a biochemical and pharmacological characterization of the venom of H. spinifer. Pharmacologically, the venom (0.125 microL/mL) produced a marked, reversible contracture in the chick biventer cervicis muscle that was blocked by d-tubocurarine (2 microM) but not by tetrodotoxin (5 microM) and omega-conotoxin GVIA (3 microM). The anticholinesterase neostigmine (1 microM) potentiated the contracture by 5.3-fold. An ultra-filtrate fraction of MW < 3000 (F3K) of the venom produced a similar contracture in the biventer muscle, whereas the retentate of MW > 3000 did not. In the rat anococcygeus muscle, the venom produced a contractile response that was partially (37.4 +/- 1.6%) blocked by atropine (5 microM); phentolamine (5 microM) blocked the remaining response. Tetrodotoxin (5 microM) did not block the contractile response of the venom on the anococcygeus muscle. Electrospray ionization-mass spectrometry/mass spectrometry confirmed the presence of high concentrations of acetylcholine (79.8 +/- 1.7 microM) and norepinephrine (146.7 +/- 19.8 microM) in H. spinifer venom, which can fully account for the observed cholinergic and adrenergic effects. In contrast to scorpion venoms that selectively target neuronal ion channels in mediating transmitter release, our data show that H. spinifer venom does not possess such activity, which likely explains the apparent lack of lethality of black scorpion envenomation.