A chloroplast-encoded gene, designated chIB, has been isolated from Chlamydomonas reinhardtii, its nucleotide sequence determined, and its role in the light-independent reduction of protochlorophyllide to chlorophyllide demonstrated by gene disruption experiments. The C. reinhardtii chIB gene is similar to open reading frame 563 (orf563) of C. moewusii, and its encoded protein is a homolog of the Rhodobacter capsulatus bchB gene product that encodes one of the polypeptide components of bacterial light-independent protochlorophyllide reduction. To determine whether the chIB gene product has a similar role in light-independent protochlorophyllide reduction in this alga, a series of plasmids were constructed in which the aadA gene conferring spectinomycin resistance was inserted at three different sites within the chIB gene. The mutated chIB genes were introduced into the Chlamydomonas chloroplast genome using particle gun-mediated transformation, and homoplasmic transformants containing the disrupted chIB genes were selected on the basis of conversion to antibiotic resistance. Individual transformed strains containing chIB disruptions were grown in the dark or light, and 17 of the 18 strains examined were found to have a "yellow-in-the-dark" phenotype and to accumulate the chlorophyll biosynthetic precursor protochlorophyllide. RNA gel blot analysis of chIB gene expression in wild-type cells indicated that the gene was transcribed at low levels in both dark- and light-grown cells. The results of these studies support the involvement of the chIB gene product in light-independent protochlorophyllide reduction, and they demonstrate that, similar to its eubacterial predecessors, this green alga requires at least three components (i.e., chIN, chIL, and chIB) for light-independent protochlorophyllide reduction.