We investigated the gene encoding the beta subunit of cGMP phosphodiesterase (PDE6B) as a candidate for generalized progressive retinal atrophy (gPRA), an autosomal recessively transmitted eye disease in dogs. The PDE6B gene was isolated from a genomic library. Single-strand conformation polymorphism analysis revealed eight intronic variations in different subsets of the 14 dog breeds investigated. In addition, we identified an 8-bp insertion after codon 816 in certain Sloughi dogs. Analysis of PRA-affected and obligatory carrier Sloughis showed that this mutation cosegregates with disease status in a large pedigree. All other exchanges identified were not located in functionally relevant parts of the gene (e.g., in the splice signal consensus sites). In most dog breeds (Labrador retriever, Tibetan mastiff, dachshund, Tibetan terrier, miniature poodle, Australian cattle dog, cocker spaniel, collie, Saarloos wolfhound, Chesapeake Bay retriever, and Yorkshire terrier), PDE6B was excluded as a candidate gene for gPRA because heterozygous allele constellations were detected in diseased animals. Therefore, the PDE6B sequence variations did not segregate together with the mutation(s) causing gPRA. Direct and indirect DNA tests concerning gPRA can be offered now for a variety of different dog breeds.