Purpose: Numerous studies have shown that the presence of clinically occult disseminated tumor cells (DTC's) in the bone marrow (BM) of breast cancer patients is associated with an unfavourable clinical outcome. Immunocytochemistry (ICC) remains the gold standard for their detection. While assays based on RT-PCR are available, they have not been used for routine detection of DTC's. Methods: To assess the quality of the assay, we performed a direct comparison of DTC detection rates in a large cohort of 385 patients using both standardized ICC and real-time RT-PCR protocols. Correlation rates were assessed, and results were compared with clinical data. Results: A significant correlation between ICC and RT-PCR was observed (P < 0.01). Positivity rates were similar (both 35%) and the results of both methods agreed in 73% of cases (280/385). Conclusions: We describe a real-time RT-PCR based protocol for DTC-detection that has been specifically designed for routine clinical laboratory use. As such, RT-PCR has the potential to become an alternative testing method for BM evaluation in breast cancer patients.