Allgrove syndrome (AS), also known as “triple-A syndrome”, is a rare cause of congenital adrenal insufficiency. It is inherited in an autosomal recessive manner and is associated with achalasia, alacrima and other neurologic defects. The disease was recently linked to chromosome 12q13 in consanguineous families of European ancestry. In the present study, we investigated 4 nonconsanguineous families with documented inheritance of AS for linkage with the reported 12q13 locus. Eighteen subjects were studied; DNA was extracted from peripheral blood and amplified by PCR with primers from polymorphic sequence tagged sites (STSs) located in the chromosome 12q13 region. Two-point logarithm of odds (LOD) score analysis revealed a maximum LOD score of 1.7 for STSs D12S361 and D12S368, without any recombinants[recombination distance (θ) = 0]. Multipoint linkage analysis defined an area of estimated genetic distance less than 0.5cM (approximately 500,000 base-pairs) between STSs D12S368 and D12S96, that is most likely to contain the AS gene(s). We conclude that in Puerto Rican families with AS, this condition maps to the chromosome 12q13 locus, revealing absence of heterogeneity in a genetically distinct population. Candidate genes in the region include those that code for several of the keratin proteins, transcription factors and others.