The proliferation and differentiation programs of gut epithelial cells are expressed rapidly and perpetually along an anatomically well defined pathway. The mouse intestine thus provides an excellent in vivo model system to define the contributions of CCAAT enhancer binding protein alpha (C/EBP alpha) and related bZIP proteins to these processes. Immunocytochemical studies revealed that C/EBP alpha is produced in villus-associated enterocytes located in the duodenum and jejunum of adult mice. The protein is located in the cytoplasmic and nuclear compartments of these cells. C/EBP alpha is not detectable in proliferating and nonproliferating epithelial cells situated in small intestinal crypts nor is it evident in any gut epithelial cell lineage located in the ileum and colon. The related C/EBP beta and C/EBP delta proteins are not detectable by sensitive immunocytochemical methods in epithelial cells distributed along the duodenal-to-colonic axis. Developmental surveys indicate that C/EBP alpha is confined to postmitotic, villus-associated epithelial cells during conversion of the polyclonal intervillus epithelium to monoclonal crypts. Analyses of intestinal isografts reveal that these developmental stage-specific, lineage-specific, differentiation-dependent, and regional patterns of C/EBP alpha expression can be established and maintained in the absence of exposure to luminal contents. Transgenic mice containing nucleotides -1178 to +28 of the rat intestinal fatty acid binding protein gene (I-FABP-1178 to +28) linked to the simian virus 40 large tumor antigen (T antigen) gene express T antigen in villus-associated enterocytes. This results in reentry of enterocytes into the cell cycle and a silencing of C/EBP alpha expression without an apparent effect on the accumulation of several markers of this lineage's terminal differentiation program or on gut morphogenesis. These findings indicate that there is a relationship between expression of C/EBP alpha in enterocytes and their exit from the cell cycle and suggest that I-FABP-1178 to +28/simian virus 40 T antigen transgenic mice could provide a screening assay for examining the role of C/EBP alpha in regulating the activity of genes known to be transcribed during differentiation of this gut epithelial cell lineage.