p16, an inhibitor of cell cycle machinery, is frequently inactivated in non-small cell carcinoma of the lung (NSCCL). To clarify the significance of p16 inactivation in the progression of lung adenocarcinoma, we immunohistochemically evaluated p16 protein status and Rb, p53 and cyclin D1 expression in 51 surgically resected adenocarcinomas that were less than 3 cm in diameter (median follow-up period: 52.5 months). Twenty-one of 51 adenocarcinomas showed negative immunostaining for p16. Twenty adenocarcinomas were also negative for Rb, while 31 and 13 were positive for p53 and cyclin D1, respectively. Loss of p16 expression was significantly correlated with scar grade, lymphatic permeation, lymph node metastasis and clinical stage. Rb protein expression was also inversely correlated with scar grade, pleural involvement and vascular invasion. When the cases were stratified according to the expression of both proteins, the Rb-/p16- subset (7/51) consisted of poorly differentiated adenocarcinoma with a higher grade of invasion. While Rb, p53 and cyclin D1 protein status showed no significant correlations with prognosis, p16 inactivation was significantly correlated with poor prognosis, and the prognosis of Rb-/p16- was the worst among the 4 subsets. Inactivation of p16 may play a role in accelerating scar formation and lymph node metastasis, and may contribute through these mechanisms to poor prognosis in patients with small-sized lung adenocarcinoma.