The aims of this study were (1) to develop sensitive and reproducible quantitative RT–PCR (QRT–PCR) assays for PVA, SCCA, and CK17 mRNAs for detecting and quantifying metastases in SLNs of HNSCC; (2) to investigate whether macrometastates (MAs) and MIs, including those sized <450 μm, can be distinguished from isolated tumour cells (ITCs) and negative nodes using these assays; and (3) to identify the most accurate marker for molecular analysis of SLNs, compared with an extensive histopathological examination of serial sections. The gene discussed is KRT17; the disease is head and neck squamous cell carcinoma.