Ferredoxin-dependent glutamate synthase (Fd-GOGAT) plays a major role in photorespiration in Arabidopsis, as has been determined by the characterization of mutants deficient in Fd-GOGAT enzyme activity (gls). Despite genetic evidence for a single Fd-GOGAT locus and gene, we discovered that Arabidopsis contains two expressed genes for Fd-GOGAT (GLU1 and GLU2). Physical and genetic mapping of the gls1 locus and GLU genes indicates that GLU1 is linked to the gls1 locus, whereas GLU2 maps to a different chromosome. Contrasting patterns of GLU1 and GLU2 expression explain why a mutation in only one of the two genes for Fd-GOGAT leads to a photorespiratory phenotype in the gls1 mutants. GLU1 mRNA was expressed at the highest levels in leaves, and its mRNA levels were specifically induced by light or sucrose. In contrast, GLU2 mRNA was expressed at lower constitutive levels in leaves and preferentially accumulated in roots. Although these results suggest a major role for GLU1 in photorespiration, the sucrose induction of GLU1 mRNA in leaves also suggests a role in primary nitrogen assimilation. This possibility is supported by the finding that chlorophyll levels of a gls mutant are significantly lower than those of the wild type when grown under conditions that suppress photorespiration. Both the mutant analysis and gene regulation studies suggest that GLU1 plays a major role in photorespiration and also plays a role in primary nitrogen assimilation in leaves, whereas the GLU2 gene may play a major role in primary nitrogen assimilation in roots.