The green alga Chlamydomonas reinhardtii has a network of fermentation pathways that become active when cells acclimate to anoxia. Hydrogenase activity is an important component of this metabolism, and we have compared metabolic and regulatory responses that accompany anaerobiosis in wild-type C. reinhardtii cells and a null mutant strain for the HYDEF gene (hydEF-1 mutant), which encodes an [FeFe] hydrogenase maturation protein. This mutant has no hydrogenase activity and exhibits elevated accumulation of succinate and diminished production of CO 2 relative to the parental strain during dark, anaerobic metabolism. In the absence of hydrogenase activity, increased succinate accumulation suggests that the cells activate alternative pathways for pyruvate metabolism, which contribute to NAD(P)H reoxidation, and continued glycolysis and fermentation in the absence of O 2 . Fermentative succinate production potentially proceeds via the formation of malate, and increases in the abundance of mRNAs encoding two malateforming enzymes, pyruvate carboxylase and malic enzyme, are observed in the mutant relative to the parental strain following transfer of cells from oxic to anoxic conditions. Although C. reinhardtii has a single gene encoding pyruvate carboxylase, it has six genes encoding putative malic enzymes. Only one of the malic enzyme genes, MME4, shows a dramatic increase in expression (mRNA abundance) in the hydEF-1 mutant during anaerobiosis. Furthermore, there are marked increases in transcripts encoding fumarase and fumarate reductase, enzymes putatively required to convert malate to succinate. These results illustrate the marked metabolic flexibility of C. reinhardtii and contribute to the development of an informed model of anaerobic metabolism in this and potentially other algae.Chlamydomonas reinhardtii is a unicellular, soil-dwelling, photosynthetic green alga that has a diversity of fermentation pathways, inferred from the full genome sequence (1, 2). It uses these pathways for ATP production during anoxia, catabolizing starch and other intracellular carbon substrates into the predominant fermentation products formate, acetate, ethanol, CO 2 , and molecular hydrogen (H 2 ) in what is classified as heterofermentation (2-7). These metabolic pathways would be active primarily at night when high rates of respiration and the absence of photosynthetic O 2 evolution cause the rapid establishment of anoxia (8), especially in soil environments with high concentrations of microbes. Moreover, C. reinhardtii can balance the use of the tricarboxylic acid cycle with fermentation when the rate of respiratory O 2 consumption exceeds the rate of photosynthetic O 2 evolution (3, 4, 9). The catabolism of intracellular carbon stores during anoxic acclimation is a key component of C. reinhardtii metabolism as this alga does not appear to effectively assimilate extracellular sugars. Acquiring a better understanding of cellular metabolisms in algae such as C. reinhardtii under various conditions will facilitate the development o...