Chlamydomonas reinhardtii chloroplasts catalyzed two sequential steps of Chl biosynthesis, S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase and Mg-protoporphyrin IX monomethyl ester oxidative cyclase. A double mutant strain of C. reinhardtii was constructed which has a cell wall deficiency and is unable to form chlorophyll in the dark. Dark-grown cells were disrupted with a BioNeb nebulizer under conditions which lysed the plasma membrane but not the chloroplast envelope. Chloroplasts were purified by Percoll density gradient centrifugation. The purified chloroplasts were used to define components required for the biosynthesis of Mg-2,4-divinylpheoporphyrin a 5 (divinyl protochlorophyllide) from Mg-protoporphyrin IX. Product formation requires the addition of Mg-protoporphyrin IX, the substrate for S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase which produces Mg-protoporphyrin IX monomethyl ester. The Mg-protoporphyrin IX monomethyl ester that is generated in situ is the substrate for Mg-protoporphyrin IX monomethyl ester oxidative cyclase. The reaction product was identified as Mg-2,4-divinylpheoporphyrin a 5 (divinyl protochlorophyllide) by excitation and emission spectrofluorometry and HPLC on ion-paired reverse-phase and polyethylene columns. Mg-2,4-divinylpheoporphyrin a 5 formation by the coupled enzyme system required O2 and was stimulated by the addition of NADP+, an NADPH regenerating system, and S-adenosyl-L-methionine. Product was formed at a relatively steady rate for at least 60 min.