This study examined the induction of the defence-related hormones jasmonic acid (JA), salicylic acid (SA) and abscisic acid (ABA) and the phytoalexin medicarpin in Medicago truncatula when challenged by the pea aphid Acyrthosiphon pisum. There was some induction of hormones in the compatible interaction between A. pisum clone N116 and M. truncatula cultivar DZA315, whereas JA, SA and medicarpin exhibited more significant increases in foliage concentration during the incompatible interaction between A. pisum clone PS01 and M. truncatula cultivar Jemalong A17. Foliar concentration of JA, SA and medicarpin exhibited a positive relationship with aphid density after 3-day feeding, whereas ABA was not affected by the presence of aphids. When aphids were restricted to a single leaf using plastic tubes, JA, SA and medicarpin displayed strong local induction, whereas there were no significant systemic increases in uninfested leaves. Medicarpin and SA appeared to increase with duration of aphid feeding, whereas JA showed a more transient increase in concentration 24 h after challenge commenced. Results suggest that increases in JA, SA and medicarpin are associated with M. truncatula resistance to particular clones of A. pisum. The variation in concentration of the defence-related compounds recorded with regard to aphid density, duration of challenge, genotypes of plant and aphids, and between locally challenged and distant leaves reinforces the need for consideration of these experimental factors when generalizing about the plant defence processes that occur during aphid-plant interactions.