Cytosine (C)-rich DNA can adopt i-motif folds tinder acidic conditions, with the human telomere i-motif providing a well-studied example. The dimensions of this i-motif are appropriate for capture in the nanocavity of the alpha-hemolysin (alpha-HL) protein pore under an electrophoretic force. Interrogation of the current vs time (i-t) traces when the i-motif interacts with alpha-HL identified characteristic signals that were pH dependent. These features were evaluated from pH 5.0 to 7.2, a region surrounding the transition pH of the i-motif (6.1). When the i-motif without polynucleotide tails was studied at pH 5.0; the folded structure entered the nanocavity of alpha-HL from either the top or bottom face to yield characteristic current patterns. Addition of a 5' 25-mer poly-2'-deoxyadensosine tail allowed capture of the i-motif from the unfolded terminus, and this was used to analyze the pH dependency of unfolding. At pH values below the transition point, only folded strands were observed, and when the pH was increased above the transition pH, the number of folded events decreased, while the unfolded events increased. At pH 6.8 and 7.2 4% and 2% of the strands were still folded, respectively. The lifetimes for the folded States at pH 6.8 and 7.2 were 21 and 9 ms, respectively, at 160 mV electrophoretic force. These lifetimes are sufficiently long to affect enzymes operating on DNA. Furthermore, these transient lifetimes are readily obtained using the alpha-HL nanopore, a feature that is not easily achievable by other methods.