We cloned a 6.0-kb HindIII fragment from the cyanobacterium Plectonema boryanum using the chloroplast chlB (ORF513) gene of the liverwort (Marchantia polymorpha) as a probe. An open reading frame (ORF508) encoding a polypeptide of 508 amino acid residues was found within the nucleotide sequence of the 4,437-bp HindIII-EcoRV subfragment. The deduced amino acid sequence of ORF508 shows very high similarity to that encoded by the liverwort chlB gene (72.7%). A mutant, YFB14, in which ORF508 was inactivated by the insertion of a kanamycin-resistance cartridge, was unable to synthesize chlorophyll, accumulating protochlorophyllide in darkness while synthesizing chlorophyll normally in the light. Thus, the chlB gene is the third gene that is essential for the light-independent reduction of protochlorophyllide. The other two genes are chlL and chlN, and the results suggest that the light-independent protochlorophyllide reductase consists of at least three subunits, which are encoded by chlL, chlN and chlB. Using an antiserum prepared against a ChlB-6xHis fusion protein expressed in Escherichia coli, we detected a protein with an apparent molecular weight of 58,000 in the membrane fraction of the cyanobacterium. These results indicate that either the cytoplasmic or thylakoid membranes could be the site of the light-independent reduction of protochlorophyllide.