Ubiquitously expressed focal adhesion kinase (FAK), linked to multiple intracellular signaling pathways, has previously been shown to control cell motility, invasion, proliferation, and survival. Using mice with a keratinocyte-restricted deletion of fak (FAKK5 KO), we report here a novel role for FAK: maintenance of adult epidermal permeability barrier homeostasis. Abundant lacunae of unprocessed lipids in stratum corneum (SC) of FAKK5 KO mice and delayed barrier recovery pointed to malfunction of pH-dependent enzymes active in extracellular space of SC. Measuring the SC pH gradient showed significantly more neutral pH values in FAKK5 KO mice, suggesting the importance of FAK for acidification. Moreover, normal functions were restored when FAKK5 KO mice were exposed to a surface pH typical of mouse SC (pH = 5.5). Baseline levels and response to barrier disruption of secretory phospholipase A2 isoforms, enzymes that mediate generation of free fatty acids in epidermis, appeared similar in both FAK K5 KO and control littermates. We found that the critical SC acidification regulator Na+/H+ exchanger 1 failed to localize to the plasma membrane in FAK-deficient keratinocytes both in vivo and in vitro. Thus, for plasma membrane localization in terminally differentiated keratinocytes, Na+/H+ exchanger 1 requires an intact actin cytoskeleton, which is impaired in FAK-deficlent cells. Copyright © American Society for Investigative Pathology.