To identify genes that are differentially expressed in human esophageal squamous cell carcinoma (ESCC), we have developed a cDNA microarray representing 34 176 clones to analyse gene expression profiles in ESCC. A total of 77 genes (including 31 novel genes) were downregulated, and 15 genes (including one novel gene) were upregulated in cancer tissues compared with their normal counterparts. Immunohistochemistry and Northern blot analysis were carried out to verify the cDNA microarray results. It was revealed that genes involved in squamous cell differentiation were coordinately downregulated, including annexin I, small proline-rich proteins (SPRRs), calcium-binding S100 proteins (S100A8, S100A9), transglutaminase (TGM3), cytokeratins (KRT4, KRT13), gut-enriched Krupple-like factor (GKLF) and cystatin A. Interestingly, most of the downregulated genes encoded Ca2+-binding or -modulating proteins that constitute the cell envelope (CE). Moreover, genes associated with invasion or proliferation were upregulated, including genes such as fibronectin, secreted protein acidic and rich in cystein (SPARC), cathepsin B and KRT17. Functional analysis of the alteration in the expression of GKLF suggested that GKLF might be able to regulate the expression of SPRR1A, SPRR2A and KRT4 in ESCC. This study provides new insights into the role of squamous cell differentiation-associated genes in ESCC initiation and progression.