Recently, we isolated a new src family member from a rat small intestinal cDNA library which by RNase protection analysis is selectively expressed in the columnar epithelium of gut. Complete nucleotide sequencing of the gastrointestinal associated tyrosine kinase (gtk) has revealed that it is a rat homologue of frk/rak-a fyn related human tyrosine kinase. Unlike frk/rak, gtk is myristylated, in vivo. Furthermore, by immunohistochemical analysis, the kinase is concentrated in the brush border membranes of epithelial cells, throughout the maturation axis of the adult small intestine. In vitro analysis revealed that gtk kinase activity is present in intestinal cells throughout their maturation, suggesting that the enzyme might influence signal transduction pathways in both mitotic and post-mitotic states. Gtk is expressed in all regions of the gastrointestinal tract which contain columnar epithelium, but is absent in the stratified epithelium of the esophagus. Moreover, during gestation, the kinase dramatically appears at high levels in plasma membranes, at the time of transition of gut cells from an undifferentiated to a simple columnar phenotype. After solubilization of cellular membranes with Triton X-100, sucrose gradient analysis of gtk revealed that it partitions differently than c-yes, demonstrating that the brush border src kinases associate with different components of the plasma membranes. These findings suggest that gtk plays a specialized role in the growth/differentiation of gut columnar epithelial cells.