Bulge structure is one of the well-known and important nucleic acid secondary structures containing unpaired nucleotides. Although the thermodynamic property has been thoroughly investigated, the detailed kinetic property of the bulged helix formation is still unknown. We now investigated the helix formation mechanism for bulged helices using temperature-jump experiments. The activation energy for the duplex association (Ea+1) obtained from the temperature dependence of the rate constants showed that the Ea+1 value depended on both the bulged nucleotide and its flanking base pairs. The activation energy for duplex dissociation (Ea-1), however, did not always depend on the bulged nucleotide. In the case of d(TAGCGTTATAA)/d(ATCCAATATT) with one C bulge (GCG-bulge helix) and d(TAGAGTTATAA)/d(ATCCAATATT) with One A bulge (GAG-bulge helix), that had different bulged nucleotides and the same flanking base-pairs, the Ea+1 value of -13.5 kcal/mol for the GCG-bulge helix was 11.9 kcal/mol mure negative than the value of -1.6 kcal/mol for the GAG-bulge helix. The Ea-1 value of 50.9 kcal/mol for the GCG-bulge helix was, however, close to 48.7 kcal/mol of the GAG-bulge helix. These data indicate that the rate-limiting steps for both the GCG-bulge and GAG-bulge helices are likely to be the same step. Furthermore, since it was known that fully matched helix formations can be estimated using nearest-neighbor parameters [Williams, A. P.; Longfellow, C. E.; Freier, S. M.; Kierzek, R.; Turner, D. H Biochemistry 1989, 28, 4283-4291], the kinetic data for a bulged helix were also analyzed and found analogous to the fully matched double helices. As a result, we found that the energy diagrams of the helix formations for bulged helices could be estimated using nearest-neighbor parameters for the DNA/DNA helix. According to these diagrams, the rate-limiting step for bulged helices can be considered to be the formation of four or five base pairs containing one bulged nucleotide. These results showed that the analysis of the kinetic behavior using nearest-neighbor parameters is a feasible approach and makes possible the understanding and prediction of folding in the unpaired regions of nucleic acids.