Promoter hypermethylation is an alternative way to inactivate tumor suppressor genes in cancer. Alterations of chromosome 3p are frequently involved in many types of cancer, including esophageal squamous cell carcinoma. Here, we investigated the methylation status and loss of heterozygosity (LOH) of 3p tumor suppressor genes. We examined the promoter methylation status of von Hippel-Lindau disease (VHL), retinoic acid receptor beta (RAR-beta), RAS association domain family 1A (RASSF1A), and fragile histidine triad (FHIT) genes in 22 esophageal squamous cell carcinoma cell lines and 47 primary tumors and corresponding noncancerous tissues by a methylation-specific PCR. In addition, we analyzed 47 paired samples for LOH at eight loci on chromosome 3p. Hypermethylation in VHL, RAR-beta, RASSF1A, and FHIT was detected in 36, 73, 73, and 50% of tumor cell lines, respectively. In primary tumors, hypermethylation in VHL, RAR-beta, RASSF1A, and FHIT was detected in 13, 55, 51, and 45%, respectively. In corresponding noncancerous tissues, hypermethylation in RAR-beta and FHIT was frequently detected in 38 and 30%, respectively, whereas no VHL hypermethylation and only 4% of RASSF1A hypermethylation were detected. Furthermore, in clinical stages I and II, hypermethylation in RAR-beta (67%) and FHIT (78%) was frequently detected, whereas no VHL hypermethylation and 11% of RASSF1A hypermethylation were detected. On the other hand, the correlation between FHIT hypermethylation and LOH at FHIT region was statistically significant (P = 0.008). Our findings suggest that hypermethylation of the RAR-beta and FHIT may play an important role in the early stage of esophageal squamous cell carcinogenesis. In addition, FHIT may be inactivated in accordance with the two-hit inactivation model, involving deletion of one allele and hypermethylation of the other.