Pleomorphic adenoma (PA) is the most common salivary gland neoplasm, and while mostly benign, recurrences (RPA) and malignant transformation to carcinoma ex-PA (CXPA) do occur. Cell cycle proteins important in its tumorigenesis have been studied as markers for PA with a high risk of RPA or CXPA. The aim of the present study was to investigate cell cycle markers p-16, cyclin D1, CDK4, E2F, and retinoblastoma (Rb) in this context. Expression of p16, cyclin D1, E2F, CDK4, and Rb was studied by immunohistochemistry in 24 cases of PA, 21 of RPA, and 2 of CXPA. The presence of HPV was assessed by in situ hybridization. Immunostaining for p16 and cyclin D1 was negative or weakly positive in most cases of PA while strongly positive in the majority of RPA and both CXPA cases. Staining for Rb and CDK4 was either negative or weakly positive in PA, RPA, and CXPA. Expression of E2F was stronger in RPA and CXPA than in PA. Nuclear reactivity for HPV was not observed in any case. In conclusion, the strong staining for p16, cyclinD1, and E2F in RPA and CXPA, while weak or negative in PA, suggests that these proteins might be involved in recurrence and malignant transformation of PA.4673 295301