There is evidence that the "acid mantle" of the stratum corneum is important for both permeability barrier formation and cutaneous antimicrobial defense. The origin of the acidic pH of the stratum corneum remains conjectural, however. Both passive (e.g., eccrine/sebaceous secretions, proteolytic) and active (e.g., proton pumps) mechanisms have been proposed. We assessed here whether the free fatty acid pool, which is derived from phospholipase-mediated hydrolysis of phospholipids during cornification, contributes to stratum corneum acidification and function. Topical applications of two chemically unrelated secretory phospholipase sPLA2 inhibitors, bromphenacylbromide and 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol, for 3 d produced an increase in the pH of murine skin surface that was paralleled not only by a permeability barrier abnormality but also altered stratum corneum integrity (number of strippings required to break the barrier) and decreased stratum corneum cohesion (protein weight removed per stripping). Not only stratum corneum pH but also all of the functional abnormalities normalized when either palmitic, stearic, or linoleic acids were coapplied with the inhibitors. Moreover, exposure of intact murine stratum corneum to a neutral pH for as little as 3 h produced comparable abnormalities in stratum corneum integrity and cohesion, and further amplified the inhibitor-induced functional alterations. Furthermore, short-term applications of an acidic pH buffer to inhibitor-treated skin also reversed the abnormalities in stratum corneum integrity and cohesion, despite the ongoing decrease in free fatty acid levels. Finally, the secretory-phospholipase-inhibitor-induced alterations in integrity/cohesion were in accordance with premature dissolution of desmosomes, demonstrated both by electron microscopy and by reduced desmoglein 1 levels in the stratum corneum (shown by immunofluorescence staining and visualized by confocal microscopy). Together, these results demonstrate: (i) the importance of phospholipid-to-free-fatty-acid processing for normal stratum corneum acidification; and (ii) the potentially important role of this pathway not only for barrier homeostasis but also for the dual functions of stratum corneum integrity and cohesion.