Friedreich ataxia (FRDA), an autosomal recessive, neurodegenerative disease is the most common inherited ataxia. The vast majority of patients are homozygous for an abnormal expansion of a polymorphic GAA triplet repeat in the first intron of the X25 gene, which encodes a mitochondrial protein, frataxin. Cellular degeneration in FRDA may be caused by mitochondrial dysfunction, possibly due to abnormal iron accumulation, as observed in yeast cells deficient for a frataxin homologue. Using RNase protection assays, we have shown that patients homozygous for the expansion have a marked deficiency of mature X25 mRNA. The mechanism(s) by which the intronic GAA triplet expansion results in this reduction of X25 mRNA is presently unknown. No evidence was found for abnormal splicing of the expanded intron 1. Using cloned repeat sequences from FRDA patients, we show that the GAA repeat per se interferes with in vitro transcription in a length-dependent manner, with both prokaryotic and eukaryotic enzymes. This interference was most pronounced in the physiological orientation of transcription, when synthesis of the GAA-rich transcript was attempted. These results are consistent with the observed negative correlation between triplet-repeat length and the age at onset of disease. Using in vitro chemical probing strategies, we also show that the GAA triplet repeat adopts an unusual DNA structure, demonstrated by hyperreactivity to osmium tetroxide, hydroxylamine, and diethyl pyrocarbonate. These results raise the possibility that the GAA triplet-repeat expansion may result in an unusual yet stable DNA structure that interferes with transcription, ultimately leading to a cellular deficiency of frataxin.