Two mollusc-specific neurotoxic peptides from the venom of the molluscivorous snail Conus pennaceus are described. These new toxins block acetylcholine receptors (AChR) of cultured Aplysia neurons. Bath application of 0.5-1 microM toxin induces 5-10-mV membrane depolarization, which recovers to the control level within 1-3 min in the presence of the toxin. This response is blocked by 1 mM hexamethonium. Concomitantly with the transient depolarization, the toxins block approximately 90% of the depolarizing responses evoked by brief iontophoretic application of acetylcholine. The pharmacology and amino acid sequences of the toxins (alpha PnIA, GCCSLPPCAANNPDYC-NH2; alpha PnIB, GCCSLPPCALSNPDYC-NH2) enable their classification as novel alpha-conotoxins. The sequences differ from those of previously described alpha-conotoxins in a number of features, the most striking of which is the presence of a single negatively charged residue in the C-terminal loop. This loop contains a positively charged residue in piscivorous venom alpha-conotoxins. In contrast to other alpha-conotoxins, which are selective for vertebrate skeletal muscle nicotinic ACh receptors, these Conus pennaceus toxins block neuronal ACh receptors in molluscs. As such they are new probes which can be used to define subtypes of ACh receptors, and they should be useful tools in the study of structure-function relationships in ACh receptors.