The car1 gene of the filamentous fungus Podospora anserina was cloned by complementation of a mutant defective for caryogamy (nuclear fusion), a process required for sexual sporulation. This gene encodes a protein that shows similarity to the mammalian PAF1 protein (Zellweger syndrome). Besides sequence similarity, the two proteins share a transmembrane domain and the same type of zinc finger motif. A combination of molecular, physiological, genetical, and ultrastructural approaches gave evidence that the P. anserina car1 protein is actually a peroxisomal protein. This study shows that peroxisomes are required at a specific stage of sexual development, at least in P. anserina, and that a functional homolog of the PAF1 gene is present in a lower eucaryote.