Electrophysiological and pharmacological techniques have been used to determine the mechanism of action of AF3 (AVPGVLRFamide) and AF4 (GDVPGVLRFamide) on the somatic musculature of two parasitic nematodes, Ascaris suum and Ascaridia galli. This has been compared to the action of acetylcholine (ACh), the excitatory transmitter at the neuromuscular junction of both nematodes. AF3 and AF4 contracted A. suum muscle with EC₅₀S of 24±6 nM (n=6) and 37±2 nM (n=6), respectively. The muscle cells were also depolarized by both peptides with EC₅₀S of 681±329 nM (AF3; n=7) and 901±229 nM (AF4; n=5). Although AF3 and AF4 elicited contractions in A. suum at a lower concentration in comparison to ACh, the maximum tension increases were significantly less, being 48±4% (AF3; P < 0.05) and 36±9% (AF4; P < 0.01) of the maximum ACh response. The ionic dependency of the depolarizations in response to AF3 and AF4 in A. suum muscle cells were compared to those of ACh. The depolarization elicited by ACh was reduced to a greater extent by reducing extracellular sodium concentration than the response to AF3 and AF4. The calcium channel blocker cobalt was more effective at blocking the AF3 and AF4 depolarizations than those to ACh. These experiments showed that the peptide responses have a greater dependency on extracellular calcium than sodium ions, in contrast to the responses to ACh. Furthermore, depolarizations in response to submaximal ACh were abolished by the nicotinic receptor antagonist mecamylamine but the responses to AF3 and AF4 were not (111±7% and 108±17% respectively, compared to control; n=6). These observations suggest that the responses to AF3 and AF4 in A. suum muscle are mediated via a mechanism independent of the A. suum nicotinic receptor. ACh, AF3 and AF4 were also found to contract and depolarize A. galli somatic muscle in a concentration dependent manner. Therefore the response to these peptides is not unique to A. suum and they may subserve a functional role in the motor nervous system of parasitic nematodes.