Polymorphonuclear neutrophil (PMN) migration across epithelia is a common feature of active inflammation. Given the suggested role of carbohydrates in this process, we examined the receptor CD44. The standard CD44 isoform was expressed at the cell surface of PMN. PMN migration across model polarized intestinal epithelia was reduced (by 60%) if the CD44 receptor was activated by either a specific antibody (clone IM7) or the natural soluble ligand, hyaluronic acid. This inhibitory effect following receptor activation occurred with both basolateral-to-apical- and apical-to-basolateral-directed migration. The anti-CD44 antibody similarly reduced PMN migration through filters in the absence of epithelia, while preincubation of the antibody with the epithelium did not alter subsequent PMN transepithelial migration. These data suggest that PMN, rather than epithelial, CD44 is responsible for these effects. A similar inhibitory effect of anti-CD44 antibody was also observed on migration of intraepithelial lymphocytes. The molecular mechanism involved in such negative signaling following CD44 activation may include modulation of outside-in cell signaling. While neither the anti-CD44 antibody nor CD44 ligand affected PMN mobilization of intracellular Ca²⁺, both led to increased adenylate cyclase activity, an inhibitory signal for PMN migration. Together, these results suggest that CD44 of PMN may potentially serve as a negative regulator of leukocyte migration across biological surfaces such as columnar epithelia.