Guanylin, an endogenous ligand for the Escherichia coli heat-stable enterotoxin receptor, is a recently characterized intestinal peptide. To understand the possible physiologic function of guanylin, we examined the cellular location of guanylin mRNA expression in the rat intestine. Intestinal cells were sequentially isolated from villous tip to crypt in rat jejunum and ileum. Northern blots of total RNA identified a single 0.65 kb guanylin transcript predominantly in the villous cell fractions. In situ hybridization studies demonstrated maximal signal intensity in villous cells in rat ileum and surface epithelial cells in the colon. In the ileum, the signal was nonuniform in distribution in the surface epithelial cells, with focal areas of intense signal in clusters of columnar absorptive cells. In both colon and ileum, signal intensity was near background level in deep crypt cells, lamina propria, and muscularis. © 1993 Academic Press, Inc.