DNA sequences associated with the mouse pink-eyed unstable mutation were identified in the absence of closely linked molecular markers and without prior knowledge of the encoded gene product. This was accomplished by "genome scanning," a technique in which high-resolution Southern blots of genomic DNAs were hybridized to a dispersed and moderately repetitive DNA sequence. In this assay, pink-eyed unstable DNA was distinguished from the DNA of wild-type and revertant mice by enhanced hybridization to one of several hundred resolved fragments. The fragment showing enhanced hybridization in pink-eyed unstable DNA was cloned and found to lie within a DNA duplication that is located close to, or within, the pink-eyed dilution locus. The duplication associated with the mouse pink-eyed unstable mutation may mediate the high reversion frequency characteristic of this mutation.