The alpha-fetoprotein (AFP) gene is transcribed in most epithelial cells lining the fetal mouse small intestine, but transcription persists in only a subset of enteroendocrine cells representing less than 1% of the total intestinal epithelial cells in the adult. The decrease in AFP expression after birth is mediated in part by a repressor element lying between -838 and -250 bp of the AFP gene. Deletion of this element from AFP minigene constructs results in high-level minigene expression in the intestines of adult transgenic mice. Although high levels of AFP minigene RNA are expressed, the fetal pattern of expression is not maintained upon deletion of the repressor element. Instead, the number of cells in which the minigene is expressed increases from less than 1% to approximately 10% of the epithelial cells in the adult small intestine, and includes the majority of the goblet cells in addition to the enteroendocrine cells. In contrast, the pattern of AFP minigene expression in the enterocytes is unaffected by deletion of the repressor element and continues to decrease in the neonate. These studies indicate that the identified AFP repressor is active specifically in goblet cells. The decrease in AFP expression in the enterocytes may be mediated by a separate cis-acting element that is contained in the AFP minigene construct. Alternatively, it is possible that mature enterocytes lack some of the positive factors required for initiation and maintenance of minigene transcription in the absence of the identified negative element.