Under anoxic conditions the green alga Chlamydomonas reinhardtii activates various 67 fermentation pathways leading to the creation of formate, acetate, ethanol and small 68 amounts of other metabolites including D-lactate and hydrogen. Progress has been 69 made in identifying the enzymes involved in these pathways and their sub-cellular 70 locations; however, the identity of the enzyme involved in reducing pyruvate to D-71 lactate has remained unclear. Based on sequence comparisons, enzyme activity 72 measurements, X-ray crystallography, biochemical fractionation and analysis of 73 knock-down mutants we conclude that pyruvate reduction in the chloroplast is 74 catalysed by a tetrameric NAD⁺-dependent D-lactate dehydrogenase encoded by 75 Cre07.g324550. Its expression during aerobic growth supports a possible function as a 76 ‘lactate valve’ for the export of lactate to the mitochondrion for oxidation by 77 cytochrome-dependent D-lactate dehydrogenases and by glycolate dehydrogenase. 78 We also present a revised spatial model of fermentation based on our 79 immunochemical detection of the likely pyruvate decarboxylase, PDC3, in the 80 cytoplasm.