Amplification of genomic DNA can confer a selective advantage in tumors by increasing the dosage of gene(s) involved in tumor development or progression. We have used a two-dimensional restriction landmark genomic scanning technique to identify gene amplification events in esophageal adenocarcinomas. We cloned a multicopy DNA fragment from a tumor two-dimensional gel and confirmed genomic amplification encompassing this fragment by Southern blot analysis. The corresponding DNA sequence was searched by BLAST, which allowed the use of an electronic polymerase chain reaction to map the amplicon to 19q12. We then characterized the amplicon using sequence-tagged site amplification mapping, an approach recently developed in our laboratory. DNAs from 65 esophageal and 11 gastric cardia adenocarcinomas and their normal controls were investigated using 11 sequence-tagged site markers neighboring the cloned fragment. The amplicon, spanning 8 centimorgans, was narrowed to a minimal region of 0.8 cM, which includes the cyclin E gene. We assayed 14 expressed sequence tags covering the minimal region for gene overexpression. Both DNA amplification and messenger RNA overexpression were observed in 7 of the 14 expressed sequence tags selected. Among them, cyclin E demonstrated the highest frequency of gene amplification and overexpression in the tumors examined. After analyzing the sequence-tagged site amplification patterns within BAC contig sequences located at 19q12 in the databases, we further defined the core amplified domain to a region 300 kilobases long. We observed amplification of 19q12 in 13.8% of esophageal adenocarcinomas. Our study is the first to map the core amplified domain of the 19q12 amplicon physically to a 300-kb region, and the data indicate that cyclin E is the probable target gene selected by the amplification event at 19q12.