AbstractCyclotides are small cysteine‐rich plant peptides similar in size and processing to the defensins. Long‐term growth of the Rubiaceae family plant Oldenlandia affinis under different conditions reveals a diverse cyclotide gene and peptide expression profile, including tissue specificity, suggesting that different cyclotides are regulated differently both spatially and in response to the environment. To determine whether cyclotide precursor gene regulation was dynamic we exposed O. affinis to a range of abiotic, biotic, and hormonal stimuli and monitored Oak1‐4 expression over a 48‐h period. Unlike some defensins, the genes for cyclotide precursor proteins Oak1‐4 did not display dynamic change, indicating that they contribute to the basal defense of O. affinis. Despite this lack of dynamism, the cyclotide profile of plants grown on plates differed markedly from field‐grown plants and so prompted attempts to discover novel cyclotides and precursor genes. The two most abundant cyclotides from plate‐grown O. affinis were sequenced and one was found to be an unusual linear cyclotide derivative, kalata B20‐lin. Degenerate PCR of plate‐grown O. affinis obtained five novel cyclotide genes including Oak9 which encodes for kalata B20‐lin and appears to have arisen by the presence of a premature stop codon. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 635–646, 2010.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com