Solubilization of senescent thylakoids from rape cotyledons in the presence of Triton X-100 was employed to establish an in vitro system that allowed the assessment of enzymatic conversion of phaeophorbide a into an uncoloured fluorescent chlorophyll catabolite, Bn-FCC-2. The action of the putative dioxygenase responsible for the cleavage of the porphyrin macrocycle depends on reduced ferredoxin as reductant. Apart from this thylakoidal catalyst, stromal protein is also required for the production of FCC-2 in vitro. The cleavage reaction does not occur with phaeophorbide b as substrate. Saturation kinetics with phaeophorbide a as substrate yielded an apparent Km-value of c. 200 μ. The enzyme contains iron as suggested by inhibitory effects of appropriate chelators. Enzyme activity lost upon treatment with bipyridyl was partly restored in the presence of Fe-ions; other metal ions such as Cu, Zn and Co were ineffective. The enzyme is absent in the thylakoids of mature green cotyledons. It appears upon the induction of foliar senescence and reaches the highest levels towards the end of the yellowing process.