The epidermal permeability barrier forms late in gestation, coincident with decreased lipid synthesis, increased lipid processing, and development of a mature, multi-layered stratum corneum. Prior studies have shown that changes in the epidermal Ca++ gradient in vivo regulate lamellar body secretion and lipid synthesis, and modulations in extracellular Ca++ in vitro also regulate keratinocyte differentiation. We asked here whether a Ca++ gradient forms in fetal epidermis in utero, and whether its emergence correlates with key developmental milestones of barrier formation and stratum corneum development. Using either ion precipitation or proton induced X-ray emission analysis of fetal mouse and rat skin, we showed that a Ca++ gradient is not present at gestational days 16-18, prior to barrier formation, and that a gradient forms coincident with the emergence of barrier competence (day 19, mouse; day 20, rat) prior to birth. These results are consistent with a role for Ca++ in the regulation of key metabolic events leading to barrier formation. Whether the calcium gradient is formed actively or passively remains to be determined.