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Ribulose-1,5-bisphosphate carboxylasey oxygenase (EC 18.104.22.168) is the key photosynthetic enzyme that catalyzes the first step of CO2 fixation. The chloroplastlocalized holoenzyme of plants and green algae contains eight nuclear-encoded small subunits and eight chloroplastencoded large subunits. Although much has been learned about the enzyme active site that resides within each large subunit, it has been difficult to assess the role of eukaryotic small subunits in holoenzyme function and expression. Small subunits are coded by a family of genes, precluding genetic screening or nuclear transformation approaches for the recovery of small-subunit mutants. In this study, the two small-subunit genes of the green alga Chlamydomonas reinhardtii were eliminated during random insertional mutagenesis. The photosynthesis-deficient deletion mutant can be complemented with either of the two wild-type small-subunit genes or with a chimeric gene that contains features of both. Thus, either small subunit is sufficient for holoenzyme assembly and function. In the absence of small subunits, expression of chloroplast-encoded large subunits appears to be inhibited at the level of translation.