Retinoblastoma (Rb) tumor suppressor genes' products and of the proteins regulating its phosphorylation and function in G1 arrest, p16INK4A and cyclin D1, play important roles in the regulation of the cell cycle. Rb gene inactivation, reflected by the absence of Rb protein expression, has been reported in oral squamous cell carcinomas. p16INK4A is frequently deleted, methylated, or mutated, and cyclin D1 gene amplification in many malignancies including oral squamous cell carcinomas (SCC). These findings suggested that Rb pathway of G1 arrest are the most commonly affected genes in Oral SCC. However, alternation of Rb pathway in salivary gland tumors was not clear. In this study, the expressions of Rb, p16INK4A, and cyclin D1 alternations were analyzed by immunohistochemical assay in 5 specimens of normal salivary glands and twenty-two cases of adenoid cystic carcinomas (ACC). Proliferating cell nuclear antigen (PCNA) labelling index (P.I.) was used for the evaluation of cell proliferation. Rb was consistently expressed in normal salivary glands and ACC. Loss of p16INK4A expression was observed in three cases (13.6%) of ACC. And overexpression of cyclin D1 was observed in four cases (18.2%). The three p16INK4A absent cases were the tumors with predominantly solid pattern and those cases were overexpressed cyclin D1. The cell proliferation activities of p16INK4A absent tumors (P.I. = 24.2 +/- 2.1%) were significantly higher than those of p16INK4A present tumors (P.I. = 10.4 +/- 3.5%) (P < 0.05). Cyclin D1 expression was also related to cell proliferation (P.I. of cyclin D1 negative cases vs. cyclin D1 positive: 10.1 +/- 3.0% vs. 22.6 +/- 3.4%) (P < 0.05). These findings suggested, however, alternations of Rb pathway were infrequent events in ACC of salivary glands and inactivation of p16INK4A, cyclin D1 overexpression may be related to the high cell proliferating activity of ACC.