The protein, p16, the product of cyclin-dependent kinase number 2 (CDKN2) gene, is one of the negative regulators of the cell cycle. CDK4, encoded by CDK4 gene, is the substrate of p16. We investigated the expression of p16 and CDK4 and their role in the progression of oral premalignant lesions (OPLs) and oral squamous cell carcinomas (OSCCs) in a Chinese cohort. A total of 74 samples were obtained from patients with hyperkeratosis (10), OPLs [30; mild (10), moderate (10) and severe (10) dysplastic lesions], OSCCs (15; 8 non-metastatic, 7 metastatic), and normal oral tissues (10), together with local lymph nodes (9) of patients with metastatic OSCCs. A labelled streptavidin biotin (LSAB) immunohistochemistry assay was performed and a semi-quantitative method was used to evaluate the staining intensity. The staining patterns of p16 and CDK4 were similar in all tissues and were located in both the nuclei and the cytoplasm. However, the antigen distribution characteristics and the degree of expression of both p16 and CDK4 varied at different developmental stages of the lesions. Hyperkeratotic and dysplastic lesions tended to display a progressively increasing degree of p16- and CDK4-positive nuclei as compared with normal tissues. Also, positive staining cytoplasm was highly evident in OSCCs with a very low nuclear staining (P<0.05). Forty-six of 56-, p16-positive cases were CDK4-positive, while only 6 were CDK4-positive but p16-negative, implying a high correlation between these parameters (r=0.813, P<0.001). This study indicates that the expression of p16 and CDK4 is intimately involved in the development of OPLs and OSCCs and the likely existence of a positive feedback loop between p16 and CDK4.