In an in vitro study to examine whether corneocytes stimulate neutrophils to cause a respiratory burst, we found that stratum corneum (SC) homogenates obtained from the sole of healthy individuals induced a substantial respiratory burst in human neutrophils when assessed by lucigenin-dependent chemiluminescence (CL) in the presence of the fresh human serum. Electron microscopically, the interaction between corneocytes and neutrophils was shown as distinctive deformation of the neutrophils adhering to the surface of the corneocytes that suggested a specific binding between SC and neutrophils. In contrast, in the heat-inactivated serum-supplemented system, the peak intensity of SC-induced CL was significantly decreased, being only slightly higher than that noted in the SC-free background. To circumvent the time-consuming preparation of the SC homogenates, we measured CL with a sheet of SC obtained by stripping with adhesive cellophane tape. In this case we used plasma because the complement was easily activated by cellophane tape itself when serum was used. To evaluate the influence of the location of horny tissue in the SC on CL in neutrophils, we used SC sheets stripped with cellophane tape from various levels of the SC. However, there was no significant difference in CL responses between SC sheets obtained by 1, 2, 3, 5, 10, or 15 strippings. Our findings suggest that when SC comes in contact with serum, it is opsonized by C3b, and such SC causes a respiratory burst of neutrophils following their specific binding on the surface of corneocytes.