Atopic dermatitis (AD) is a chronic inflammatory disease associated with changes in stratum corneum (SC) structure and function. The breakdown of epidermal barrier function in AD is associated with changes in corneocyte size and maturation, desquamation, lipid profiles, and some protease activities. The purpose of this study was: (i) to examine physiological changes in lesional (L) skin of acute eczematous AD, compared with nonlesional (NL) AD skin and healthy (H) skin, using sequential tewametry and SC protein analysis to estimate SC thickness; and (ii) to assess which serine proteases might be involved in pathogenesis. Six subjects with H skin, six AD patients with NL skin and six AD patients with mild to moderate eczema (L skin) were enrolled. Skin was assessed using several noninvasive techniques but SC thickness was estimated using tewametry and SC protein content of D-Squame strippings. SC integrity was determined by sequential tape stripping (D-Squame) and infrared densitometry. Kallikreins, plasmin, urokinase and leucocyte elastase protease activities together with a novel SC tryptase-like enzyme activity were quantified. Transepidermal water loss (TEWL) levels after D-Squame stripping were elevated in L compared with NL and H skin at all sampling points (P < 0.05). Conversely, the amount of SC removed by sequential tape stripping was decreased in L skin, indicating increased intracorneocyte cohesion (P < 0.05). By correlating 1/TEWL values and SC removed as an estimate of SC thickness, a significantly thinner SC was observed in L compared with NL and H skin (P < 0.05). Elevated extractable serine protease activity was measured in AD skin in the order: SC tryptase-like enzyme (45x), plasmin (30x), urokinase (7.1x), trypsin-like kallikreins (5.8x) and chymotrypsin-like kallikreins (3.9x). Leucocyte elastase activity was not detected in H and NL skin but was observed in AD SC samples (L skin). All enzymes were elevated in the deeper layers of L SC compared with NL and H SC samples. All consistently elevated SC protease activities were significantly correlated with the bioinstrumental data. We report increased serine protease activities in acute eczematous AD, especially in deeper layers of the SC, including SC tryptase-like enzyme, plasmin, urokinase and leucocyte elastase activities. These elevations in protease activities were associated with impaired barrier function, irritation, and reduced skin capacitance. Increased SC cohesion was apparent despite elevated TEWL during tape stripping, which would indicate reduced SC thickness in acute eczematous lesions of AD. Indeed, this was observed using an estimate of SC thickness.