Two critical defensive functions of the outer epidermis, the permeability barrier and antimicrobial defense, share certain structural and biochemical features. Moreover, three antimicrobial peptides (AMPs), i.e., mouse Β-defensin 3 (mBD3), mouse cathelicidin antimicrobial peptide (mCAMP), and the neuroendocrine peptide, catestatin (Cst), all localize to the outer epidermis, and both mBD3 and mCAMP are secreted from the epidermal lamellar bodies with other organelle contents that subserve the permeability barrier. These three AMPs are upregulated in response to acute permeability barrier disruption, whereas conversely, mCAMP-/- mice (unable to combat Gram-positive pathogens) also display abnormal barrier homeostasis. To determine further whether these two functions are co-regulated, we investigated changes in immunostaining for these three AMPs in skin samples in which the permeability barrier function in mice had been either compromised or enhanced. Compromised or enhanced barrier function correlated with reduced or enhanced immunohistochemical expression of mCAMP, respectively, but conversely with Cst expression, likely due to the role of this AMP as an endogenous inhibitor of cathelicidin expression. mBD3 expression correlated with experimental barrier perturbations, but poorly with developmental changes in barrier function. These studies show that changes in cathelicidin and Cst expression parallel changes in permeability barrier status, with a less clear relationship with mBD3 expression. © 2011 The Society for Investigative Dermatology.