The retinoblastoma (Rb) pathway controls the G1-S checkpoint of the cell cycle. Inactivating mutations and deletions of p16 and Rb and up-regulation of cyclin D1 disrupt this pathway and occur in many cancers. However, the concurrent expression of these genes in primary and metastatic gastric cancer is unknown, and the prognostic value of their expression is unclear. In this study, the expression of cyclin D1, retinoblastoma protein (pRb), and p16 in 67 resected gastric adenocarcinomas, and of pRb and p16 in 40 associated lymph node metastases, was determined using a streptavidin-biotin-peroxidase immunohistochemical method. Relationships with clinical and pathological features were analyzed. Cyclin D1 overexpression (>/=5% expression) was seen in 55% of cancers; pRb loss (<20% expression), in 33%; p16 loss (<10% expression), in 49%; and at least 1 of these abnormalities, in 92.5%. Cyclin D1 overexpression was associated with poor differentiation (P = 0.027) and signet ring cell type (P = 0.029). pRb expression was lower in lymph node metastases than in the corresponding primary tumors (P <0.001). Univariate and multivariate survival analysis (minimum follow-up 72 months or until death) revealed that <20% pRb expression, <30% pRb expression, and International Union Against Cancer stage >2 were associated with worse overall survival. The results suggest that Rb pathway disturbances play an important role in gastric carcinogenesis. The poor prognosis of cancers with low pRb expression and the reduced pRb expression in lymph node metastases raise the possibility that Rb and related genes also influence progression.