Mitochondrial citrate synthase (mCS) is the initial enzyme of the tricarboxylic acid (TCA) cycle. Despite the key position of this protein in respiratory metabolism, very few studies have addressed the question of the effects of the absence of mCS in development. Here we report on the characterization of 15 point mutations and a complete deletion of the cit1 gene, which encodes mCS in the filamentous fungus Podospora anserina. This gene was identified genetically through a systematic search for suppressors of the metabolic defect of the peroxisomal pex2 mutants. The cit1 mutant strains exhibit no visible vegetative defects. However, they display an unexpected developmental phenotype: in homozygous crosses, cit1 mutations impair meiosis progression beyond the diffuse stage, a key stage of meiotic prophase. Enzyme assays, immunofluorescence and western blotting experiments show that the presence of the mCS protein is more important for completion of meiosis than its well-known enzyme activity. Combined with observations made in budding yeast, our data suggest that there is a general metabolic checkpoint at the diffuse stage in eukaryotes.