Useful markers showing biological aggressiveness of head and neck squamous cell carcinoma (HNSCC) are needed to predict the outcome of the disease. MMP-2 is associated with aggressive behavior of several solid cancers. In this study, the clinical significance of tumor tissue and circulating immunoreactive proteins for MMP-2 and TIMP-2 was assessed in HNSCC. The study group consisted of 74 patients with HNSCC and 44 healthy controls. Expression of MMP-2 and TIMP-2 was examined in paraffin-embedded tumor sections by immunohistochemical methods using specific antibodies. The pretreatment serum levels of MMP-2, TIMP-2 and MMP-2:TIMP-2 complex were quantitatively measured by ELISA assay. The results were compared with the clinicopathological factors of the disease and the patients' outcome. Immunohistochemical overexpression of MMP-2 in tumor was found to be prognostic for shortened survival in HNSCC, the 5-year cumulative relapse-free survival being 42% in patients with high positivity for MMP-2 in tumor vs 61% in cases with a negative or only weakly MMP-2-positive tumor (P=0.045). Tissue MMP-2 positivity was also strongly connected with later lymph node or hematogenic relapses and associated to the cause-specific survival (P=0.055). Similarly, the 5-year cause-specific survival was significantly poorer in patients with extensive positive immunostaining for tumor TIMP-2 than in those with a TIMP-2-negative tumor (40 vs 64%, P=0.038). Patients with a TIMP-2-positive tumor also had an unfavorable 5-year relapse-free survival rate (43 vs 60%, respectively, P=0.071). Additionally, the overexpression of TIMP-2 was a powerful predictor of later lymph node or hematogenous metastases in HNSCC. Serum levels of MMP-2, TIMP-2 or MMP-2:TIMP-2 complex failed to associate with the clinical behavior of HNSCC in this material. The results of this study provide evidence that MMP-2 and TIMP-2 immunoreactive protein in tumor tissue of HNSCC patients, but not when assayed from preoperative serum samples, are prognostic in estimation of the aggressive clinical course of HNSCC.