Antisense oligonucleotides (AOs) can facilitate the expression of internally deleted dystrophin in dystrophin-deficient Duchenne muscular dystrophy (DMD) by correcting the reading frame of the pre-mRNA with AO-mediated exon skipping. An antisense 18-mer 2'-O-methyl RNA/ethylene-bridged nucleic acid chimera AO targeting exon 45 of the dystrophin gene, AO85, can induce exon 45 skipping efficiently in cultured cells. AO85 is expected to facilitate dystrophin expression in 8%-9% of all DMD patients. Here, we examined the kinetics of AO85-mediated exon 45 skipping in a cell-free splicing system. In vitro transcribed pre-mRNAs containing dystrophin exon 45 and part of its flanking introns within a hybrid minigene were incubated with HeLa cell nuclear extract, and the resultant mRNAs were amplified by semiquantitative reverse transcriptase-polymerase chain reaction. Time-course analysis revealed that the splicing process fitted well to first order kinetics. Addition of AO85 produced an extra spliced product, deleting exon 45 (Δexon 45), indicating AO85-mediated exon 45 skipping. Production of Δexon 45 increased linearly with increasing concentrations of AO85, reaching a maximum of nearly 80% of the transcripts. The half-maximal effective concentration (EC(50)) of AO85 was 58.0 nM. The percentage of Δexon 45 among the transcripts decreased inversely with the pre-mRNA concentration; Lineweaver-Burk plotting revealed a competitive fashion of AO85 action. The low EC(50) indicates high potential of AO85 for clinical application.