Cyclin D1 and p16^INK4A are molecules with pivotal roles in cell cycle control and the development of diverse human cancers, and overexpression of cyclin D1 and loss of p16^INK4A expression are common genetic events in head and neck squamous cell carcinoma. The prognostic significance of these molecular events at different sites within the head and neck, however, remains controversial. Thus, we sought to determine the relationship between cyclin D1 and/or p16^INK4A expression and disease outcome in squamous cell carcinoma of the anterior tongue. Immunohistochemical detection of nuclear proteins cyclin D1, p53, and p16^INK4A, and the Ki-67 labeling index was undertaken in tissue sections from 148 tongue cancers treated by surgical resection. Nuclear antigen status was analyzed in relation to pathological variables, tumor recurrence, and patient survival. Statistical significance was assessed using χ² analysis for pathological variables and the Kaplan-Meier method, log rank test, and the Cox proportional hazards model for survival parameters. Overexpression of cyclin D1 occurred in 68% of tumors (100 of 147) and was associated with increased lymph node stage (P = 0.014), increased tumor grade (P = 0.003), and reduced disease-free (P = 0.006) and overall (P = 0.01) survival. Loss of p16^INK4A expression was demonstrated in 55% of tumors (78 of 143) and was associated with reduced disease-free (P = 0.007) and overall (P = 0.014) survival. Multivariate analysis confirmed that in addition to pathological stage and regional lymph node status, cyclin D1 overexpression and loss of p16^INK4A expression are independent predictors of death from tongue cancer. Loss of p16^INK4A in the presence of cyclin D1 overexpression conferred a significantly worse disease-free (P = 0.011) and overall (P = 0.002) survival at 5 years. p53 nuclear accumulation and the Ki-67 labeling index were not prognostic. These data indicate that cyclin D1 overexpression and loss of p16^INK4A expression predict early relapse and reduced survival in squamous cell carcinoma of the anterior tongue. Simultaneous assessment of cyclin D1 and p16^INK4A protein levels define subgroups of patients at increased risk of relapse and may be of clinical utility in optimizing therapy.