Previous studies have established that two Xbal polymorphic restriction fragments [11.5 and 44 kilobases (kb)] hydridizing to a full length debrisoquin hydroxylase cDNA are associated with two different mutant alleles for the debrisoquin hydroxylase gene (IID6). An independent allele, defined by Xbal 16- and 9-kb fragments, has previously been identified only in extensive metabolizers, precluding determination of whether this restriction fragment length polymorphism (RFLP) pattern identifies a functional or nonfunctional mutant allele of IID6. We phenotyped and performed RFLP studies in the family of a poor metabolizer (PM) propositus with the Xbal 16 + 9 allele. Three family members were of the PM phenotype, two of whom had the 16 + 9 allele, indicating that this RFLP pattern identified a nonfunctional IID6 allele in this family. Moreover, additional RFLP analyses indicated that the PM mother had two different IID6 mutant alleles that produce the Xbal 29-kb fragment, indistinguishable from the RFLP pattern produced by a functional IID6 allele. These data indicate that the Xbal 16 + 9 RFLP pattern can identify a mutant IID6 allele and that at least two different nonfunctional mutant alleles can produce the Xbal 29-kb fragment found in all extensive metabolizers.