Outer dense fibers are a major constituent of the sperm tail and outer dense fiber 2 (ODF2) protein is one of their major components. ODF2 shares partial homology with cenexin 1 and cenexin 2, regarded as centriolar proteins. We show that ODF2 and cenexin 2 transcripts are the product of differential splicing of a single gene, designated Cenexin/ODF2 and that cenexin 1 is an incomplete clone of ODF2. ODF2 terminates in exon 20b whereas in cenexin 2 this exon is spliced out and translation terminates in exon 24. We demonstrate a transcriptional switch during rat testicular development, from somatic-type to testis-type ODF2 and cenexin transcripts during the onset of meiosis. The switch is completed when spermiogenesis is established. ODF2 immunoreactive sites were visualized in the acroplaxome, along the sperm tail and the centrosome-derived sperm head-to-tail coupling apparatus. An unexpected finding was the presence of ODF2 antigenic sites, but not cenexin antigenic sites, in the dense fibrillar component of the nucleolus of Sertoli cells, spermatogonia and primary spermatocytes. The characterization of the genomic origin, processing and developmental expression of ODF2 transcript isoforms and their protein products can help reconcile differences in the literature on the role of ODF2 and cenexin in the centrosome. Furthermore, the finding of ODF2 in the dense fibrillar component of the nucleolus suggests that this protein, in addition to its presence in sperm outer dense fibers and centrosome, highlights and adds to the nucleolar function during spermatogenesis and early embryogenesis.