The properties of simple trinucleotide repeats generate increased interest as expansions of certain trinucleotide blocks cause human diseases. Here, we studied protein binding and structural features of a perfect (gaa.ttc) 2 4 tract in its original genomic environment. Electrophoretic mobility shift assays revealed that HeLa nuclear proteins bind to the DNA fragment containing the (gaa.ttc) 2 4 block. Competition experiments using simple (gt.ac) n repeats differing in length and flanking regions showed no cross-reactivity with the major retarded band. For the specific (gaa.ttc) n /protein complex, a binding constant of 9.3x10 - 9 mol/l was determined. DNase I footprinting revealed protein binding sites located exclusively within the repeat with a preference for the (gaa) 2 4 strand. OsO 4 and DEPC modifications followed by electrophoretic and electron microscopical analyses showed that the (gaa.ttc) 2 4 block forms different types of intramolecular triple helices: Under superhelical stress, different *H-DNA isomers are evident, whereas exclusively H-Y forms were detected in the relaxed state. Together, these data have functional implications for genomic (gaa.ttc) n tracts.