During intestinal disease induced by Salmonella typhimurium transepithelial migration of neutrophils (PMN) rapidly follows attachment of the bacteria to the epithelial apical membrane. Among the events stimulated by these interactions is the release of chemotaxins that guide PMN through the subepithelial matrix and subsequently through the epithelium itself (McCormick, B.A., S.P. Colgan, C. Delp-Archer, S.I. Miller, and J.L. Madara. 1993. J. Cell Biol. 123:895-907). Given the substantial volume flow that normally characterizes matrix compartments underlying transporting epithelia, it is unclear how such transmatrix signaling is sustained. Here we show that when underlying matrices are isolated from biophysically confluent polarized monolayers of the human intestinal epithelial cell line T84, they fail to support substantial transmatrix migration of PMN unless an exogenous chemotactic gradient is imposed. In contrast, such matrices isolated from confluent monolayers apically colonized with S. typhimurium support spontaneous transmatrix migration of PMN. Such chemotactic imprinting of underlying matrices is resistant to volume wash and is paralleled by secretion of the known matrix-binding chemokine IL-8. Chemotactic imprinting of the matrix underlying S. typhimurium-colonized monolayers is dependent on epithelial protein synthesis, is directional implying the existence of a gradient, and is neutralized by antibodies either to IL-8 or to the IL-8 receptor on PMN. An avirulent S. typhimurium strain, PhoPc, which attaches to epithelial cells as efficiently as wild-type S. typhimurium, fails to induce basolateral secretion of IL-8 and likewise fails to imprint matrices. Together, these observations show that the epithelial surface can respond to the presence of a luminal pathogen and subsequently imprint the subepithelial matrix with retained IL-8 gradients sufficient to resist washout effects of the volume flow that normally traverses this compartment. Such data further support the notion that the primary role for basolateral secretion of IL-8 by the intestinal and likely other epithelia is recruitment of PMN through the matrix to the subepithelial space, rather than directing the final movement of PMN across the epithelium.