Pardaxin, an excitatory neurotoxin, is a new tool for studying the machinery of neurotransmitter secretion. At noncytotoxic concentrations (< 1 x 10(-5) M), pardaxin stimulated exocytosis, as assessed by the concomitant release of catecholamines, ATP and dopamine-beta-hydroxylase from bovine adrenal medullary chromaffin cells in the presence or absence of extracellular calcium. At higher concentrations (> 2 x 10(-5) M), pardaxin was increasingly cytotoxic, as inferred from trypan blue uptake, release of lactate dehydrogenase and 51Cr (ED50 = 100 microM). The role of intracellular calcium ([Ca++]i) homeostasis in pardaxin action was investigated by using the fluorescent calcium indicator Fura-2. In the presence of extracellular calcium, addition of noncytotoxic concentrations of pardaxin yielded a steady, concentration-dependent rise in [Ca++]i (ED50 = 1 microM). Depolarization of chromaffin cells by high K+ reduced pardaxin binding and abolished the pardaxin-evoked rise in [Ca++]i. In the absence of extracellular calcium, pardaxin failed to elicit an elevation of [Ca++]i. These data suggest that, in the presence of extracellular calcium, pardaxin might cause elevations in [Ca++]i and neurotransmitter release, concomitant with inducing transmembranal Ca++ influx. However, the complex concentration dependence of [Ca++]i and the fact that pardaxin stimulated secretion without a rise of [Ca++]i suggest that the toxin, in addition to being a pore-forming molecule, might directly affect exocytosis in a Ca(++)-independent way. In proposing a pharmacological working model, we hypothesize that pardaxin might present a molecular structure which mimics an essential step in the endogenous docking mechanism between secretory granules and the plasma membrane.