Early systemic spread of occult tumor cells that may develop into founders of incurable distant metastasis has been identified in prostate cancer patients by reverse transcription-PCR (RT-PCR) amplification of prostate-specific antigen (PSA) mRNA. Nevertheless, the introduction of this new staging tool into the clinical setting has been hampered by the disparate and contradictory data on the sensitivity and specificity of RT-PCR methods reported recently. We used PSA RT-PCR to examine the influence of analytical variables such as priming and enzyme of reverse transcriptase reaction, temperature and time of primer annealing, primer extension and denaturation, as well as the concentrations of magnesium chloride, Taq polymerase, deoxynucleotide triphosphate, primers and BSA on the amplification process. By systematically varying these chemical and physical components, we could demonstrate a significant increase in amplification yield and in stringency of primer annealing. This may explain the wide variety of published findings on molecular staging of prostate cancer, which currently impedes the clinical introduction of PSA RT-PCR assays in prostate cancer. Methodological analyses are needed for standardization and quality assurance to achieve reproducible molecular methods that can be used in clinical practice.