Our discovery that plasmids containing the Friedreich's ataxia (FRDA) expanded GAA.TTC sequence, which forms sticky DNA, are prone to form dimers compared with monomers in vivo is the basis of an intracellular assay in Escherichia coli for this unusual DNA conformation. Sticky DNA is a single long GAA.GAA.TTC triplex formed in plasmids harboring a pair of long GAA.TTC repeat tracts in the direct repeat orientation. This requirement is fulfilled by either plasmid dimers of DNAs with a single trinucleotide repeat sequence tract or by monomeric DNAs containing a pair of direct repeat GAA.TTC sequences. DNAs harboring a single GAA.TTC repeat are unable to form this type of triplex conformation. An excellent correlation was observed between the ability of a plasmid to adopt the sticky triplex conformation as assayed in vitro and its propensity to form plasmid dimers relative to monomers in vivo. The variables measured that strongly influenced these measurements are as follows: length of the GAA.TTC insert; the extent of periodic interruptions within the repeat sequence; the orientation of the repeat inserts; and the in vivo negative supercoil density. Nitrogen mustard cross-linking studies on a family of GAA.TTC-containing plasmids showed the presence of sticky DNA in vivo and, thus, serves as an important bridge between the in vitro and in vivo determinations. Biochemical genetic studies on FRDA containing DNAs grown in recA or nucleotide excision repair or ruv-deficient cells showed that the in vivo properties of sticky DNA play an important role in the monomer-dimer-sticky DNA intracellular intercon-versions. Thus, the sticky DNA triplex exists and functions in living cells, strengthening the likelihood of its role in the etiology of FRDA.