Many plant species produce phytoecdysteroids (PEs: i.e. analogues of insect steroid hormones). There is increasing evidence that PEs are used as a chemical defence by plants against non-adapted insects and nematodes. PEs are good candidates for the development of an environmentally safe approach to crop protection. Most crop species do not accumulate PEs. However, many arguments support the idea that most, if not all, plant species have the genetic ability to produce PEs, but the biosynthetic pathway is not active. A better understanding of the PE biosynthetic pathway and its regulation is consequently necessary. Spinach is one of the very few crop plants which produce large amounts of PEs, of which 20-hydroxyecdysone is the major component. Labeling experiments with radiolabeled precursor (mevalonic acid), putative ecdysteroid intermediates and 20-hydroxyecdysone itself have allowed investigation of PE biosynthesis and transport during spinach development. Biosynthesis takes place in older leaf sets ("sources"), but not in the young developing ones, which in contrast accumulate (acting as "sinks") the PEs produced by the older leaves. PEs are thus continuously redistributed within the developing plant, as its leaf set number increases. The biosynthetic pathway has been analyzed using excised leaves and various labeled precursors, and a preferential sequence of the last steps has been established. Although they do not produce PEs, apical leaf sets are nevertheless able to perform several putative terminal steps of PE biosynthesis. The regulatory mechanisms of PE synthesis appear to involve a direct negative feedback of 20-hydroxyecdysone (the major PE in spinach) on its own synthesis; thus, a sustained synthesis in older leaves requires that they can export the PE they produce.