The nature and length of loops connecting cytosine tracts in i-motif structures may affect their stability. In this work, the influence of the nature of the nucleobases located in two of the loops of an intramolecular i-motif is studied using spectroscopy, separation techniques, and multivariate data analysis. Insertion of bases other than thymine induces an additional acid-base equilibrium with pKa~4.5. The presence of two guanine bases in the loops, placed opposite to each other, decreases the thermal stability of the structure. In contrast, thymine and cytosine bases in these positions stabilize the structure