Several members of the Amy-2 alpha-amylase multigene family from the CE/J strain of mouse have been cloned in cosmid vectors. Structural analysis of these recombinants reveals that the cloned Amy-2 copies contain tandem 5' termini. The duplicated 5'-terminal elements, which lie upstream from the Amy-2 cap site, are separated from their Amy-2 homologues by about 8000 bases. The orientation of these 5' orphons is the same as that of Amy-2. Gene titration and cloning experiments suggest that at least four of the approximately 15 Amy-2 copies present in the CE/J genome contain 5' orphon elements. The extent of sequence homology between 5' orphons and their gene homologues has been determined by DNA sequence analysis. All the orphons are identical and contain the entire 185 base-pairs of the first exon, 49 base-pairs of the first intron and more than 400 base-pairs of the Amy-2 5' flanking region. Intron and flanking-region sequences of the orphons differ by about 20% from their Amy-2 counterparts, and the exon by about 8%. The TATA box and the cap site are conserved, while the ATG translation initiation signal is mutated to ATA in the orphon. No transcription initiation has been detected at the orphon cap site using run-off transcription in isolated pancreatic nuclei in vitro.