Trefoil peptides are members of a unique family of proteins found predominately throughout the gastrointestinal tract, whose proposed functions include mucus stabilization, stimulation and/or differentiation of epithelial cells during wound repair. Recent trefoil knockout studies have reported delays in epithelial cell migration or maturation pathways together with almost a complete lack of mucus. In order to fully explore the role of trefoil peptides in gastrointestinal maturation, these studies were undertaken to accurately characterize the expression of trefoil peptides in the developing rat gut. The results of RPA suggest that trefoil mRNA's are expressed as early as 15 days post coitus (dpc) in the intestine and stomach. Proteins are detected at 17 dpc by radioimmunoassay and immunohistochemical studies, which localize trefoil peptide expression to the lumenal surface of epithelial cells. At 17 dpc the gut is lined by pseudo-stratified, undifferentiated epithelial cells. Polarized, columnar cells are not detected until at least 18 dpc, with sparse mucus staining and parietal cell markers not being detected until 18 and 19 dpc respectively. This data demonstrates that trefoil peptides are early markers of epithelial cell maturation in the developing rat gut. The time course of their expression, well before the mucus cell type is specified, suggests a potential role in epithelial cell differentiation.