The antisense method is one of the most promising anti-cancer methods, however, the design of antisense oligonucleotides is difficult because many factors affecting their activitiy and stability must be considered. Recently, the oligonucleotide stabilities related to the antisense effects were quantitatively investigated based on nearest-neighbor parameters. We demonstrated that DG°37, hyb , a free energy change for the hybridization of antisense oligodeoxynucleotides (ODNs) with target RNAs is related to the RNase H cleavage of TAg (SV40 large T antigen) mRNA, the expression of a rabbit globin mRNA, and the protein function encoded by hMDR1 (human multidrug resistance-1) mRNA, while DG°37, hp , a free-energy change for hairpin formations of the antisense ODNs significantly affected the arrest efficiency of the DHFR (dihydrofolate reductase) mRNA transcription, the expression of the pr??1(I) chain of human, and the hybridization extent for HIV-1 a-1. For ras RNA (Ha-ras mRNA), DG°37, sc , a free energy change for the conformational change of the mRNA required for antisense ODN binding showed the best correlation with the equilibrium constants for the hybridization with their target RNA. On the other hand, the antisense effects ifor the HSV-1 IE5 (herpes simplex virus type 1 immediate early pre-mRNA5) showed less of a relationship to the hybridization stability of the antisense ODNs with the target pre-mRNA, because the antisense ODNs targeting the pre-mRNA must collapse its secondary structure around the splicing site to cancel out the expected antisense effects. Based on these results, we illustrate a new concept for the design of antisense ODNs based on deltaG 37, hyb , deltaG 37, hp , and deltaG 37, sc .