To determine the role of the cysteine proteinase inhibitor cystatin C in the invasive behavior of squamous cell carcinoma of the head and neck (SCCHN), Cystatin C protein level was measured in 82 pairs of primary tumour tissue and adjacent noncancerous mucosa, using the enzyme-linked immunosorbent assay. The median level of cystatin C in tumour tissue was 1.18 times lower than that in corresponding mucosa (P=0.031). In normal mucosa samples, the cystatin C level was influenced by the site of sampling: it was lower in nonlaryngeal tissue samples (oral cavity, oro- or hypopharynx) than in laryngeal samples (P=0.004). The tumour cystatin C level correlated inversely with pN-stage (P=0.047), whereas a trend of lower cystatin C levels was observed in the group with extranodal tumour extension compared to those with no extranodal spread (P=0.069). In univariate analysis, the patients with low tumour cystatin C levels exhibited poor disease-free survival (DFS, P=0.013) and disease-specific survival (DSS, P=0.013). In multivariate analysis, the most powerful predictor of survival was pN-stage (DFS: P=0.040, HR 2.78; DSS: P=0.011, HR 4.36,), followed by the cystatin C level (DFS: P=0.043, HR 0.22; DSS: P=0.067, HR 0.25). When comparing the prognostic strength of cystatin C to that of stefin A, another cysteine proteinase inhibitor, which emerged as the most significant prognosticator for survival in our previous study analysing the same cohort of patients, stefin A proved to be significantly more reliable predictor for both DFS and DSS than cystatin C. Our results indicate that cystatin C is implicated in the invasive behavior of SCCHN, and that there are variations in regulation of proteolytic pathways under nonmalignant conditions, inherent to individual subsites inside the upper aerodigestive tract. The correlation between high cystatin C levels and improved survival concurs with the concept of the protective role of high levels of cysteine proteinase inhibitors in tissue homogenates that has been previously suggested by the survival results in breast and lung carcinoma as well as SCCHN.