Cyclotides, a family of disulfide-rich mini-proteins, show a wide range of biological activities, making them interesting targets for pharmaceutical and agrochemical applications, but little is known about their natural function and the events that trigger their expression. An investigation of nutritional variations and irradiation during a batch process involving plant cell cultures has been performed, using the native African medical herb, Oldenlandia affinis, as a model plant. The results demonstrated the biosynthesis of kalata B1, the main cyclotide in O. affinis, in a combined growth/nongrowth-associated pattern. The highest concentration, 0.37 mg g(-1) dry weight, was accumulated in irradiated cells at 35 mumol m(-2) s(-1). Furthermore, 12 novel cyclotides were identified and the expression of various cyclotides compared in irradiated vs non-irradiated cultures. The results indicate that cyclotide expression varies greatly depending on physiological conditions and environmental stress. Kalata B1 is the most abundant cyclotide in plant suspension cultures, which underlies its importance as a natural defense molecule. The identification of novel cyclotides in suspension cultures, compared to whole plants, indicates that there may be more novel cyclotides to be discovered and that the genetic network regulating cyclotide expression is a very sensitive system, ready to adapt to the current environmental growth condition.