1H NMR spectra of the dipiperazine‐based DNA minor groove binding compound Hoechst 32985 indicate that at room temperature and low pH (2.90) a single conformation is detected. This is attributed to the form in which both piperazine rings are protonated at the N‐methyl and the bulky methyl and aromatic groups are in an equatorial arrangement. The aliphatic regions of the spectra broaden considerably with an increase in either temperature or pH, indicating the presence of a dynamic exchange process. This process results in interchange of axial and equatorial environments of the piperazine ring protons and is attributed to combined ring and nitrogen inversions. Analysis of the variable‐temperature spectra for a solution at pH 2.90 allowed a Gibb's free energy of activation (ΔG‡) of 69.4±0.4 kJ mol−1 to be calculated for this process.