This study reports on the relationship between quantitative (99m)Tc-HYNIC radiolabelled annexin V tumour uptake measurements, Fas ligand (FasL) expression, matrix metalloproteinase-9 (MMP-9) expression, microvessel density (MVD) and the number of tumour-infiltrating lymphocytes in squamous cell carcinoma of the head and neck (SCCHN) patients. Twenty-eight patients (24 men and 4 women; mean age 59 years, range 43-83 years) suffering from a primary ( n, number of patients=22) or locally recurrent ( n=6) SCCHN were studied. All patients underwent a spiral CT scan, allowing estimation of lesion size in three dimensions, and (99m)Tc-HYNIC annexin V scintigraphy within 1 week of each other. Biopsies or resection of the suspected primary tumour or local recurrence for histopathological analysis were performed on all patients within a period of 10 days following (99m)Tc-HYNIC annexin V scintigraphy. The percentage uptake of the injected dose of (99m)Tc-HYNIC annexin V in visible tumour lesions on scintigrams divided by the tumour volume, derived from CT, was related to MVD and to histological score (HSCORE) values for MMP-9 and FasL expression as well as to the number of tumour-infiltrating lymphocytes (CD45 staining). Median percentage absolute tumour uptake of the injected dose/cm(3) tumour volume derived from tomographic images was 0.0001% (SD 0.0001%) at 5-6 h p.i. (range: 0.000007-0.0003%). Mean HSCORE for MMP-9 tumour staining was 2.1 (SD 0.84). Mean HSCORE for FasL tumour staining was 2.49 (SD 0.92). At the sites of tumour containing the highest number of vessels, the mean MVD was 20 vessels/field at the hot spot (range 1-73). The median number of tumour-infiltrating lymphocytes was 500 (range 100-5,000). The percentage absolute tumour uptake of the injected dose/cm(3) tumour volume derived from tomographic images correlated linearly with FasL HSCORES( r=0.47, P=0.02). No correlation was found between the percentage absolute tumour uptake of the injected dose/cm(3) tumour volume derived from tomographic images and MMP-9 HSCORES, MVD or the number of tumour-infiltrating lymphocytes. MVD correlated significantly with MMP-9 HSCORES ( r=0.44, P=0.03).