To understand the alterations of Rb tumor suppressor gene and the relationship between defects in the Rb and p53 pathways in human esophageal carcinogenesis, we examined the loss of heterozygosity (LOH) of the Rb gene and immunohistochemical staining of pRb protein in 56 esophageal squamous cell carcinoma specimens and related the results to the p53 gene alterations. Using four introgenic polymorphic markers as probes, we observed LOH of the Rb gene in 30 of the 55 informative tumor samples. Immunohistochemical analysis revealed different patterns of pRb expression among the tumor samples. In the 56 cases, 16 displayed extensive pRb staining comparable to that of the adjacent normal epithelia, whereas 33 showed either significantly decreased or no pRb staining and 7 had a focal staining pattern reflecting heterogeneous cancer nests in the tumor with respect to Rb status. In the tumor samples containing Rb LOH, 90% showed low or no pRb expression, whereas in samples without Rb LOH, only 20% had altered pRb expression. There was a strong association between LOH of the Rb gene and alteration of pRb expression in our samples (P < 0.0001), suggesting LOH is a main event leading to Rb inactivation. We found that Rb LOH was more frequent in tumors with p53 mutations (P < 0.05), which occurred in 31 of the 49 cases analyzed. When the status of Rb and p53 alterations was evaluated by the combined results of immunohistochemical and genetic analyses, we found that alteration of Rb and p53 had an even stronger association in our esophageal squamous cell carcinoma samples (P = 0.0015). Among the 51 cases in which both the Rb and p53 status were determined, 31 contained alterations in both genes, and only 5 and 6 cases were altered in only Rb and only p53, respectively. Our results suggest that defects in the Rb and p53 pathways and their potential synergistic effect in deregulating cell cycle and apoptosis are major mechanisms for esophageal carcinogenesis.