We examined the pattern of gene expression resulting from spontaneous differentiation of Caco-2 BBe cells to gain insight into the molecular changes necessary for enterocyte differentiation. RNA was prepared from cells harvested at three cell stages: proliferating (50% confluent, 2 d in culture), post-proliferative- non-differentiated (8 d), and differentiated (15 d). Gene expression profiles were determined using Affymetrix Human Genome U95A GeneChips. Differentially expressed genes were identified following statistical analysis (i.e. ANOVA, bootstrapping adjustments to p-values, false detection rate criterion). 1150 unique genes were identified as differentially expressed; expression of 48.6% fell and 46% increased from 2 d to 15 d while 5.4% had expression that either peaked or dipped at 8 d. Genes expressed during differentiation included several small intestine specific genes involved in nutrient transport/metabolism: e.g. DCT1, hephaestin, folate receptor 1, sucrase-isomaltase, apolipoproteins CI, CIII, B100, H, and M, indicating that this colonic adenocarcinoma cell line has a hybrid colonocyte/enterocyte phenotype. Patterns of gene expression based upon functional classification suggest a role for cell-cell/cell-matrix interactions, suppression of Wnt signaling, and activation of TGF and PI3-kinase pathways during enterocyte differentiation.