These studies characterized the paralytic and neurophysiological effects of an atracotoxin (ACTX), from the Australian funnel web spider, Hadronyche versuta, and compared it to the established P/Q-type calcium channel blocker, omega-agatoxin-IVA (omega-Aga-IVA). ACTX-induced paralysis was of a spastic form in housefly ( Musca domestica) larvae, but it was inactive on neuromuscular junction of housefly and tobacco budworm ( Heliothis virescens). On cockroach ( Periplaneta americana) cercal nerve-giant fiber synapse preparations, both toxins were effective blockers with potencies in the nanomolar range, but some spontaneous, high frequency trains of action potentials were observed with ACTX. In Drosophila melanogaster central nervous preparations, blockage of nerve firing occurred within 20 min when the nerve sheath was intact, demonstrating that the barrier could be breached by ACTX in vitro. There was a potent (pM) excitatory response to ACTX in this tissue, prior to the onset of block at higher concentrations. In contrast, omega-Aga-IVA was a pure blocker in both cockroach and Drosophila preparations. These studies demonstrate that central synaptic calcium channels underlie the action of ACTX. ACTX-dependent neuroexcitation has a number of possible mechanisms that warrant further study.