BACKGROUND Expression of the gene encoding the neurotensin/neuromedin N (NT/N) is developmentally regulated in the gut in a distinctive temporal and spatial fashion. Src kinase, a nonreceptor tyrosine kinase, has been implicated in the growth and differentiation of various tissues; its role in gut differentiation is not known. The purpose of this study was to determine whether the Src signaling pathway plays a role in the activation of the human NT/N promoter. METHODS Caco-2 cells, a human colon cancer cell line that can differentiate to a small bowel phenotype, were transiently transfected with human NT/N promoter fragments linked to luciferase and various amounts of Src expression plasmids or dominant negative Raf; luciferase and beta-galactosidase activities were measured after 48 hours. RESULTS Cotransfection of Src resulted in an approximate eightfold increase of NT/N promoter activity; mutation of a proximal activating protein-1/cyclic adenosine monophosphate responsive element site resulted in a dramatic decrease of Src-mediated NT/N induction. Cotransfection with a dominant negative Raf plasmid partially blocked Src-mediated NT/N activation. CONCLUSIONS Src increases NT/N promoter activity in Caco-2 cells acting, in part, through a proximal AP-1/CRE promoter element. In addition, Src regulation of the NT/N promoter appears to be mediated through a Raf-dependent pathway. We propose that Src may play a role in tissue-specific gene expression in the gut.