Cyclin D1 proto-oncogene is a key regulator of the mammalian cell-cycle acting at the restriction point in late G1. Ampli®cation of the cyclin D1 locus, located on chromosome 11q13, as well as cyclin D1 protein overexpression have been reported in several human malignancies. The purpose of this study was to evaluate cyclin D1 gene copy status and protein expression during the multistep process of head and neck tumorigenesis, using a combination of¯uorescence in situ hybridization and immunohistochemistry techniques. From 29 selected patients presenting with head and neck squamous carcinoma and whose tumor cytospins had been previously screened for presence (16 cases) or absence (13 cases) of ampli®cation at the 11q13 band, we analysed 46 para n-embedded tissue specimens that demonstrated, besides the primary tumor, the presence of contiguous adjacent normal tissue and/or premalignant lesions. Of the 16 ampli®ed cases, nine demonstrated a continuous progression from premalignant to invasive carcinoma and seven (77.7%) of these cases showed cyclin D1 gene ampli®cation in premalignant lesions prior to development of invasive carcinoma. Increased cyclin D1 protein expression was observed in all 16 ampli®ed tumors and ®ve of the 13 (38.4%) nonampli®ed tumors. Interestingly, dysregulated cyclin D1 expression was also found in the premalignant lesions adjacent to all 16 ampli®ed tumors, and it appeared to precede cyclin D1 gene ampli®cation. In contrast no dysregulated expression was detected in the premalignant lesions of the non-ampli®ed tumors. In conclusion, these ®ndings provide strong evidence for early dysregulation of cyclin D1 expression during the tumorigenesis process and suggest that dysregulated increased expression precedes and possibly enables gene ampli®cation.