To overcome the disadvantages of two-round nested PCR, we developed a simple and robust closed single-tube nested PCR method (antisense PCR). The method uses antisense oligonucleotides that carry a 5′ tag and that can potentially hybridize to the 3′ ends of the outer primers, depending on the annealing temperature. During initial cycles, which are performed at a high annealing temperature, the antisense oligonucleotides do not hybridize and amplification is directed by the outer primers. During later cycles, for which the annealing temperature is decreased, the outer primers hybridize to the antisense oligonucleotides, extend to produce sequences that are mismatched to the amplicon templates, and consequently become inactivated, whereas the inner primers hybridize to the amplicon templates and continue amplification. Antisense quantitative PCR (qPCR) was compared with one-round qPCR for real-time amplification of four PCR targets (BCR, APC, N-RAS, and a rearranged IGH gene). It had equal amplification efficiency but produced much less nonspecific amplification. Antisense PCR enables both endpoint detection and real-time quantification. It can substitute for two-round nested PCRs but may also be applicable to instances of one-round PCR in which nonspecificity is a problem.