Angiogenesis is a fundamental process in tumor growth and metastasis, and its significance and that of vascular endothelial growth factor (VEGF) expression as prognostic indicators have been documented for various types of human tumors. However, the mechanisms responsible for angiogenesis in head and neck squamous cell carcinoma are not well defined. To examine the relationship between angiogenesis and the phenotypic progressions of head and neck tumorigenesis, we used immunohistochemistry to analyze VEGF expression and microvessel density in 70 paraffin-embedded specimens that contained adjacent normal epithelium, premalignant lesions, or both from 57 patients with head and neck squamous cell carcinoma. Ten samples of normal oral mucosa were obtained from people who did not smoke or drink alcohol and included in the analysis as normal controls. Microvessel density was evaluated by averaging 10 microscopic fields (x400) in a defined area of each specimen. The degree of VEGF expression was assessed on a cell-by-cell basis in 10 microscopic fields (x200) in a defined area on a scale ranging from 0 (no expression) to 3+ (highest level of expression). In addition, the weighted mean index of VEGF expression was calculated. The mean +/- SD weighted mean index of VEGF expression in normal control epithelium (1.10 +/- 0.38, n = 10) was higher than it was in adjacent normal epithelium (0.82 +/- 0.27, n = 13; P = 0.04). VEGF expression decreased as samples ranged from normal adjacent epithelium to hyperplasia (0.78 +/- 0.28, n = 21), mild dysplasia (0.70 +/- 0.29, n = 28), moderate dysplasia (0.67 +/- 0.29, n = 11), severe dysplasia (0.51 +/- 0.39, n = 6), and squamous cell carcinoma (0.20 +/- 0.27, n = 70; overall P = 0.0001). VEGF expression was two times lower in cases with nodal disease (0.17 +/- 0.26, n = 29) than it was in nonnodal disease (0.32 +/- 0.29, n = 16; P = 0.02). Microvessel density showed no significant difference from adjacent normal epithelium premalignant lesions to cancer. In tumor, no correlation was seen between VEGF expression or microvessel density and differentiation, primary tumor site, T stage, or smoking status. These findings indicate that VEGF expression is down-regulated during head and neck tumorigenesis. However, further studies are required to better understand the mechanism of VEGF down-regulation in head and neck tumorigenesis.