Cytokeratins are potential markers for epithelial cell detection in hematological tissues. Thus, we developed a nested reverse transcriptase-polymerase chain reaction (RT-PCR) strategy to detect cytokeratin 20 (CK20) mRNA and studied its sensitivity and specificity as a molecular marker of occult breast cancer cells. In cell dilution experiments with human breast cancer cell lines, the limit of detection was 1 tumor cell in 1,000 hematological cells. In RNA dilution experiments of breast cancer cells' RNA in E. Coli tRNA, the CK20 transcript was only detectable when at least 1 ng of total tumor RNA was present in a total of 1 microg of RNA mixture. In parallel experiments using colorectal cancer specimens, CK20 mRNA was detected with as little as 1 pg of total tumor RNA, suggesting a low level of CK20 mRNA expression in breast cancer cells. The CK20 transcript was detected in all six tumors and five hematological samples of breast cancer patients but in none of nine hematological cell lines. However, CK20 transcript was also detected in unfractionated nucleated cell population of hematological samples from 23 of 31 (74%) healthy volunteers and from 12 of 24 (50%) patients with hematological malignancies. When mononucleated and polymorphonucleated cell populations of hematological samples from these control groups were screened separately, CK20 expression was detected in 94% of polymorphonucleated cell fractions and in 44% of mononucleated cell subpopulations. Thus, we conclude that the low sensitivity and specificity of RT-PCR detection of CK20 mRNA limits its usefulness for breast cancer cell detection in hematological products.