The rat intestinal fatty acid binding protein (I-FABP) gene has been used as a model to study temporal and spatial differentiation of the gut epithelium while its protein product has been used as a model for examining the atomic details of noncovalent fatty acid-protein interactions. We have isolated the mouse I-FABP gene (Fabpi) and determined its nucleotide sequence. Comparisons of the orthologous mouse, rat, and human I-FABP genes revealed three conserved domains in their otherwise divergent 5′ nontranscribed sequences. RNA blot hybridization and multilabel immunocytochemical methods were used to compare the developmental stage-specific patterns of activation of the rat and mouse genes. In addition, Fabpi expression in enterocytes was examined as a function of their differentiation along the crypto-to-villus and duodenal-to-colonic axes of the small intestine. Based on the similar temporal and geographic patterns of mouse and rat I-FABP expression described here and the results of our earlier studies of transgenic mice containing rat Fabpi/human growth hormone fusion genes, we propose that one of the conserved domains, spanning nucleotides −500 to −419 in mouse Fabpi, and/or a 14-bp element, are necessary for establishing and maintaining its region-specific expression along the duodenal-to-colonic axis of the perpetually renewing gut epithelium. Finally, predictions of the structure of mouse I-FABP using the refined 2.0 Å model of rat I-FABP, suggest that a proline found at position 69 of the mouse, but not rat, protein may affect its ligand binding properties.