Retinal degeneration is inherited in an autosomal recessive pattern in the retinal degeneration (rd) mouse. The defective gene for this disease has been mapped to mouse chromosome 5 between the well-defined (anchor) genes Afp and Gus. We recently cloned a putative rd cDNA, zr.408, using a strategy based on subtractive and differential hybridization. zr.408 was shown to hybridize to a larger message from rd/rd mice than from normal mice in Northern blots; was mapped to mouse chromosome 5; and was used to detect restriction fragment length polymorphisms (RFLPs) between rd/rd and +/+ DNA in genomic Southern blots. In order to obtain further evidence that zr.408 does in fact correspond to the rd gene, we used two methods to position zr.408 on chromosome 5. Analysis of an intersubspecific backcross localized the sequences corresponding to zr.408 between the genes Afp and Gus, as expected for rd. The second approach involved an interspecific backcross using C57BL/6J-rd/rd mice congenic for the normal allele of rd, which was derived from the wild mouse, Mus spretus; the results of this study showed that zr.408 is at or near the rd locus. These two studies add evidence to the existing data, which suggest that zr.408 is the correspondent of the rd gene.