Positional cloning is a powerful strategy for identifying the site of disease-producing mutations when the underlying biochemical defect is unknown. The approach also offers new methods for the presymptomatic diagnosis of genetic disease. Using these methods we have localized the PKD1 gene, mutated in the majority of PKD1 families, to a small (500 kb) segment of chromosome 16, band p13.3. Virtually all of this interval has been cloned in cosmids and lambda bacteriophage. Over 20 sets of non-overlapping cDNA clones have been isolated from the region. Sequence and mutational analyses are currently underway. In addition, a set of polymorphic clones has been identified for presymptomatic diagnosis. Included in this set are several highly variable [CA]n microsatellite repeats. These highly informative markers can be rapidly assayed from a small amount of genomic DNA using the polymerase chain reaction. Despite these advances, presymptomatic diagnosis cannot be established with certainty in many families. However, identification of the PKD1 gene itself will eventually allow diagnosis by direct detection of mutations.