Allelic loss on chromosome 3p occurs frequently in esophageal cancer. The human ribosomal protein L14 gene (RPL14) is located on chromosome 3p21.3. In the present study, we investigated alteration of RPL14 at both the genomic DNA and RNA levels in 129 Chinese esophageal squamous cell carcinomas (ESCC) and 17 dysplasia adjacent to tumor tissues by a combination of tissue microdissection, microsatellite analysis of the intragenic marker, reverse transcriptase-polymerase chain reaction (RT-PCR) and direct sequencing. In the tested informative cases, loss of heterozygosity (LOH) of RPL14 was observed in 29 out of 68 (43%) tumors. Decreased expression of the gene was detected in 31 out of 49 (63%) carcinomas. No mutation was found in the remaining RPL14 allele of the tumors with LOH. We examined subsequently the allelic status of RPL14 in the dysplasia (preneoplastic lesions) between malignant tissues and histologically normal epithelia. Of 17 tested dysplasia in which the tumors showed LOH, eight (47%) displayed the same allelic loss as their corresponding tumors, seven (41%) exhibited microsatellite instability (MSI), and only two retained both the RPL14 alleles. The data suggest that alteration of RPL14 occurred frequently in ESCC and might be an earlier event in the tumorigenesis of the esophagus. Analysis to RPL14 gene may contribute to the early detection of ESCC as a potential molecular marker.