The whole cell variant of the patch clamp technique was used to investigate the actions of two novel insect peptides on high voltage-activated Ca 2+ currents in cultured dorsal root ganglion (DRG) neurones. The insect peptides (PMP-D2 and PMP-C) were isolated originally from insect brains and fat bodies, and have been found to have similar three-dimensional structures to the N-type Ca 2+ channel inhibitor ω-conotoxin GVIA (ω-CgTx GVIA). High voltage-activated Ca 2+ currents were activated from a holding potential of −90 mV by depolarizing step commands to 0 mV. Extracellular application of synthetic PMP-D2 or PMP-C (1 μM) attenuated high voltage-activated Ca 2+ currents. The effects of PMP-C were strongly dependent on the frequency of current activation, but inhibition was apparent and reached a steady state after 20 steps when currents were evoked for 30 msec at 0.1 Hz. The actions of the two insect peptides overlapped both with each other and with ω-CgTx GVIA, suggesting that N-type Ca 2+ current was predominantly sensitive to these peptides. Low voltage-activated T-type current and 1,4-dihydropyridine sensitive L-type Ca 2+ currents were insensitive to 1 μM PMP-D2 and PMP-C, which indicates a degree of selectivity. The presence of a fucose group on PMP-C abolished the ability of this peptide to attenuate high voltage-activated Ca 2+ currents, which may reflect a mechanism by which peptide function could be regulated in insects. The electrophysiological data are supported by studies on 45Ca 2+ influx into rat cerebrocortical synaptosomes. Both PMP-D2 (10 μM), PMP-C (10 μM) and ω-CgTx GVIA (1 μM) attenuated a proportion of 45Ca 2+ influx into the synaptosomes, but additive effects of these peptides were not observed. We conclude that these naturally occurring peptides obtained from invertebrate preparations have inhibitory effects on N-type Ca 2+ channels. Although the peptides have related three-dimensional structures, they have distinct amino acid sequences and appear to have different mechanisms of action to produce inhibition of mammalian neuronal high voltage-activated Ca 2+ channels. © 1997 Elsevier Science Ltd. All rights reserved.