Pseudoknots have diverse and important roles in many biological functions. We used a combination of UV spectroscopy and differential scanning calorimetry to investigate the effect of the loop length on the unfolding thermodynamics of three sets of DNA stem-loop motifs with the following sequences: (a) d(GCGCT(n)GCGC), where n = 3, 5, 7, 9; (b) d(CGCGCGT(4)GAAATTCGCGCGT(n)AATTTC), where n = 4, 6, and 8; and (c) d(TCTCTT(n)AAAAAAAAGAGAT(5)TTTTTTT), where n = 5, 7, 9, and 11. The increase in loop length of the first set of hairpins yielded decreasing TMs and constant unfolding enthalpies, resulting in an entropy driven decrease in the stability of the hairpin (Delta G degrees = -7.5 to -6.1 kcal/mol). In the second set, the increase in the length of the loops yielded similar TMs and slight increases in the unfolding enthalpies. This translated into more stable pseudoknots with an increasing G from -13.2 to -17.1 kcal/mol. This effect can be rationalized in terms of the increased flexibility of the pseudoknot with larger loops optimizing base-pair stacking interactions. In the last set of molecules, the increase in the length of one of the loops yielded an increase in the TMs and larger increases in the enthalpies, which stabilize the pseudoknot significantly increasing the Delta G degrees from -8.5 to -16.6 kcal/mol. In this set, the thymine loop is complementary to the stem of AT base pairs and the longer loops are able to form T*A T base triplets due to the partial folding of the thymine loop into the ceiling of the major groove of the duplex, thus yielding a net formation of 1-3 T*AT/T*AT base-triplet stacks at the middle of its stem, depending on the loop length.