The intestinal epithelium is an active participant in the mucosal immune response against luminal pathogens. Microorganisms and their cell wall products, i.e. lipopolysaccharide (LPS), can stimulate the enterocyte to produce an innate immune response with the increased production of IL-8 via an activation of the transcription factor NFkappaB. The innate response mechanism, however, has not been understood until the recent description of a family of human toll-like receptors (hTLR) on immune cells that interact with LPS and modulate the IL-8 response via an intracellular signal transduction pathway similar to that of the IL-1 receptor family. Accordingly, in this study we have sought to determine the constitutive and regulated expression of hTLR on a nonmalignant human fetal primary small intestinal cell line (H4 cells) and on small intestinal samples of ileum from human fetuses (age 18-21 wk). Specimens were examined by reverse-transcription PCR, Western blot analysis, and immunofluorescence for hTLR2 and hTLR4 mRNA and protein and to determine whether their expression was regulated by LPS or by an endogenous inflammatory stimulus, IL-1beta. hTLR2 and hTLR4 were expressed constitutively on H4 cells and on human fetal small intestinal enterocytes, predominantly on the basolateral surface of crypt enterocytes. Inflammatory stimuli appeared to regulate hTLR transcription (IL-1beta increased both hTLR2 and hTLR4 whereas LPS decreased hTLR4) and possibly translation (qualitative observations). The presence of hTLR on human fetal enterocyte suggests a mechanism for the innate immune response to pathogens and could provide the basis for further study of the accentuated inflammatory response in age-dependent gastrointestinal diseases such as necrotizing enterocolitis.