Using Affymetrix HG-U133 Plus 2.0 array and laser capture microdissection techniques, we determined whether different zones of the same pancreatic tumor exhibited differential expression of genes. Human L3.6pl pancreatic cancer cells were implanted into the pancreas of nude mice. Three weeks later when tumors were 7 to 9 mm in diameter, gene expression patterns in tumor cells within the central and peripheral zones were compared, and 1,222 genes showed statistically significant differences. Bioinformatic functional analysis revealed that 346 up-regulated genes in the peripheral zone were related to cytoskeleton organization and biogenesis, cell cycle, cell adhesion, cell motility, DNA replication, localization, integrin-mediated signaling pathway, development, morphogenesis, and IkappaB kinase/nuclear factor-kappaB cascade; 876 up-regulated genes in the central zone were related to regulation of cell proliferation, regulation of transcription, transmembrane receptor protein tyrosine kinase signaling pathways, response to stress, small GTPase-mediated signal transduction, hexose metabolism, cell death, response to external stimulus, carbohydrate metabolism, and response to wounding. The reliability of the microarray results were confirmed by in situ hybridization analysis of the expression of two genes. Collectively, the data showed zonal heterogeneity for gene expression profiles in tumors and suggest that characterization of zonal gene expression profiles is essential if microarray analyses of genetic profiles are to produce reproducible data, predict disease prognosis, and allow design of specific therapeutics.