The cyclotides are stable plant-derived mini-proteins with a topologically circular peptide backbone and a knotted arrangement of three disulfide bonds that form a cyclic cystine knot structural framework. They display a wide range of pharmaceutically important bioactivities, but their natural function is in plant defense as insecticidal agents. To determine the influence of individual residues on structure and activity in the prototypic cyclotide kalata B1, all 23 non-cysteine residues were successively replaced with alanine. The structure was generally tolerant of modification, indicating that the framework is a viable candidate for the stabilization of bioactive peptide epitopes. Remarkably, insecticidal and hemolytic activities were both dependent on a common, well defined cluster of hydrophilic residues on one face of the cyclotide. Interestingly, this cluster is separate from the membrane binding face of the cyclotides. Overall, the mutagenesis data provide an important insight into cyclotide biological activity and suggest that specific self-association, in combination with membrane binding mediates cyclotide bioactivities.Cyclotides are a fascinating family of plant-derived mini-proteins of around 30 residues that are characterized by the topologically unique combination of a head-to-tail cyclized backbone and a cystine knot. The original discovery of the prototypic member kalata B1 (1) was guided by the indigenous medicinal use in Africa of a tea made from the plant Oldenlandia affinis (Rubiaceae) to accelerate childbirth. Kalata B1 was identified as a uterotonic component of the plant, but its unusual macrocyclic nature (2) and the fact that it was just one member of a large family of related macrocyclic peptides were elucidated only recently (3). Cyclotides have now been reported in several other species of the Rubiaceae (coffee) family (4, 5) and in all tested members of the Violaceae (violet) plant family (3, 6, 7) bringing the number of known cyclotides to over 100.