The exact quantification of tiny amounts of nucleic acids in biological samples continues to remain a requirement in both the experimental and the diagnostic laboratory. Competitive PCR involves the coamplification of a target DNA sample with known amounts of a competitor DNA that shares most of the nucleotide sequence with the target; in this way, any predictable or unpredictable variable affecting PCR amplification has the same effect on both molecular species. Competitive PCR therefore permits the quantification of the absolute number of target molecules in comparison to the amount of competitor DNA. Although requiring intensive post-PCR manipulation, the accuracy of competitive PCR by far exceeds that of any other quantitative PCR procedure, including real-time PCR. This protocol covers all stages in the competitive PCR and RT-PCR methods, from the design and construction of competitor molecules, and the competitive PCR itself, to the analysis of data and quantification of target DNA. Once the correct primers are available, the protocol can be completed in about 24 h.