The thermodynamics of the stacking to unstacking transitions of 24 single-stranded DNA sequences (ssDNA), 10-12 bases in length, in sodium phosphate buffer were determined from 10 to 95 degrees C, using differential scanning calorimetry (DSC). An additional 22 ssDNA sequences did not exhibit an S<=>U transition in this temperature range. The transition properties of the ssDNA sequences with <or=60% self-complementarily in the reverse direction were independent of concentration with transition temperatures ranging from 15 to 70 degrees C, van't Hoff transition enthalpies from 92 to 201 kJ mol(-1) and transition enthalpies from 5 to 75% of the corresponding van't Hoff transition enthalpies. Since all the 16 doublets and 60 of the 64 triplets are present in both the transition and the non-transition ssDNA sequences, it is unlikely that the nucleation subset initiating stacking of the sequence is a specific doublet or triplet subset. Of the 141 quadruplet subsets of the 46 sequences, each transition ssDNA sequence contained at least one or more quadruplets not found in the non-transition ssDNA sequences. It could be concluded that the thermal stability of the stacked conformation was dependent on the presence of a possible nucleation quadruplet and the length of the ssDNA sequence and not on the G or C content of the ssDNA sequence, nor on the number of purine bases in the sequence.