In this study, the origin of the monovinyl chlorophyll a carboxylic biosynthetic route was investigated in barley (Hordeum vulgare L.) and com (Zea mays L.). Protoporphyrin IX accumulated in vivo or in vitro was found to be all of the divinyl form. Furthermore, the conversion of divinyl protoporphyrin IX to monovinyl protoporphyrin IX in vitro was not observed. In contrast, the biosynthesis and accumulation of monovinyl Mg-protoporphyrin IX and its methyl ester occurred in etiolated leaves and divinyl Mg-protoporphyrin IX was convertible to monovinyl Mg-protoporphyrin IX in vitro. These results suggest that the monovinyl chlorophyll ${alpha}$ carboxylic biosynthetic route in plants may originate from the divinyl Mg-protoporphyrin IX pool.