We developed a novel polymerase chain reaction (PCR)-based method to analyze simultaneously the relative expression of two genes in a single PCR reaction. The method, relational PCR (R-PCR), utilizes special PCR primers that enable a PCR reaction to be converted from a standard uniplex reaction to a multiplex reaction in which all products are dependent on the same reaction components for amplification. We show that the quantitative ability of R-PCR is unaffected by sample nucleic acid input concentration over a range of 25-fold (30 to 750 ng of total RNA) and demonstrate excellent interexperimental reproducibility. We used R-PCR to analyze estrogen receptor gene expression in a series of invasive breast carcinomas, and our results show an excellent correlation between estrogen receptor mRNA expression and protein product accumulation determined by standard immunocytochemistry on paraffin sections.