Serial analysis of gene expression (SAGE) provides quantitative and comprehensive expression profiling in a given cell population. In our efforts to define gene expression alterations in Barrett's-related adenocarcinomas (BA), we produced eight SAGE libraries and obtained a total of 457,894 expressed tags with 32,035 (6.9%) accounting for singleton tags. The tumor samples produced an average of 71,804 tags pur library, whereas normal samples produced an average of 42,669 tags per library. Our libraries contained 67,200 unique tags representing 16,040 known gene symbols. Five hundred and sixty-eight unique tags were differentially expressed between BAs and normal tissue samples (at least twofold; P ≤ 0.05), 395 of these matched to known genes. Interestingly, the distribution of altered genes was not uniform across the human genome. Overexpressed genes tended to cluster in well-defined hot spots located in certain chromosomes. For example, chromosome 19 had 26 overexpressed genes, of which 18 mapped to 19q13. Using the gene ontology approach for functional classification of genes, we identified several groups that are relevant to carcinogenesis. We validated the SAGE results of five representative genes (ANPEP, ECGF1, PP1201, EIF5A1, and GKN1) using quantitative real-time reverse-transcription PCR on 31 BA samples and 26 normal samples. In addition, we performed an immunohistochemistry analysis for ANPEP, which demonstrated overexpression of ANPEP in 6/7 (86%) Barrett's dysplasias and 35/65 (54%) BAs. ANPEP is a secreted protein that may have diagnostic and/or prognostic significance for Barrett's progression. The use of genomic approaches in this study provided useful information about the molecular pathobiology of BAs.