Magnesium-protoporphyrin chelatase lies at the branch point of the heme and (bacterio)chlorophyll biosynthetic pathways. In this work, the photosynthetic bacterium Rhodobacter sphaeroides has been used as a model system for the study of this reaction. The bchH and the bchI and -D genes from R. sphaeroides were expressed in Escherichia coli. When cell-free extracts from strains expressing BchH, BchI, and BchD were combined, the mixture was able to catalyze the insertion of Mg into protoporphyrin IX in an ATP-dependent manner. This was possible only when all three genes were expressed. The bchH, -I, and -D gene products are therefore assigned to the Mg chelatase step in bacteriochlorophyll biosynthesis. The mechanism of the Mg chelation reaction and the implications for chlorophyll biosynthesis in plants are discussed.