Pea albumin I b (PA I b) is a small sulphur-rich peptide from pea seeds, also called leginsulin due to the binding properties of its soybean orthologue. Its insecticidal properties were discovered more recently, By using a combination of molecular, biochemical and specific insect bioassays, we characterised new genes and their products from the seeds of four legume species. Two species (Glycine max and Phaseolus vulgaris) display most of the characteristics of the Pisum sativum type: homologous genes and predicted toxic hydrophobic peptides with similar post-translational processing. The third species (Medicago truncatula) possesses homologous genes and high insecticidal activity, but no specific biochemical detection of the peptide products was obtained, indicating possible variant post-translational processing. Our combined approach appears to be efficient for a broad study of A I b within legumes. (C) 2004 Elsevier Ireland Ltd. All rights reserved.