The properties of an intramolecular triplex formed in vitro at the 5'- flanking region of the human γ-globin genes were studied by chemical and physical probes. Chemical modifications performed with osmium tetroxide, chloroacetaldehyde, and diethyl pyrocarbonate revealed the presence of non- paired nucleotides on the 'coding strand' at positions -209 through -217. These reactivities were induced by negative supercoiling, low pH, and magnesium ions. Downstream point mutations associated with hereditary persistence of fetal hemoglobin (HPFH) altered the extent of the modifications and some of the patterns. Specifically, C-202 → G and C- 202 → T significantly decreased the reactivities, whereas the patterns were increased and altered in the T-198 → C. C-196 → T and C- 195 → G caused local decreases in reactivity. Modifications at the upstream flanking duplex were modulated by the composition of the vector sequence. In summary, our data indicates the formation of an intramolecular triplex between nucleotides -209 to -217 of the 'non-coding strand' and the downstream sequence containing the HPFH mutations. All of the HPFH point mutations altered the structure. More than one sequence alignment is possible for each of the triplexes. In addition, a consequence of some of the point mutations may be to facilitate slippage of the third strand relative to the Watson-Crick duplex.