A locus in the human genome, not associated with any specific gene, has been found to be a site of restriction fragment length polymorphism. The polymorphism was found by hybridizing a 16-kilobase-pair segment of single-copy human DNA, selected from the human genome library cloned in phage lambda CH4A, to a Southern transfer of total human DNA digested with EcoRI. DNAs from a number of individuals from within Mormon pedigrees as well as random individuals have been examined. The locus is highly variable, with at least eight alleles present, homozygotes accounting for less than 25% of the individuals examined. The polymorphism appears to be the result of DNA rearrangements rather than base-pair substitutions or modifications. Examination of the DNA from seven members of a family revealed fragment lengths that are consistent with their inheritance as Mendelian alleles through three generations.