Previous studies have demonstrated that perturbations in barrier function stimulate epidermal lipid synthesis and that this increase can be prevented by occlusive membranes. These observations suggest that epidermal lipid synthesis might be related to barrier function and raised the question whether transcutaneous water flux might regulate epidermal lipogenesis. In the present study we first abrogated the barrier with acetone, and then compared the rate of repletion of stainable lipids, barrier recovery, and epidermal lipogenesis in animals covered with occlusive membranes or vapor-permeable membranes versus uncovered animals. Acetone treatment of hairless mice removed stainable neutral lipids from the stratum corneum, with repletion evident both biochemically and histochemically within 48 hr in uncovered animals. In contrast, when the animals were covered with an occlusive membrane, the usual return of stratum corneum lipids was aborted. Since application of vapor-permeable membranes allowed normal lipid repletion, occlusion alone is not responsible for the inhibition of lipid repletion. Acetone treatment also perturbed epidermal barrier function, which returned to normal in uncovered animals in parallel with the reappearance of stratum corneum lipid. However, when animals were covered with an occlusive membrane, barrier function did not recover normally. In contrast, occlusion with vapor-permeable membranes allowed barrier function to recover normally. Finally, whereas occlusive membranes prevented the characteristic increase in epidermal lipid synthesis that follows barrier perturbation, epidermal lipid synthesis was increased in animals covered with a vapor-permeable membrane. These results point to transepidermal water flux itself as the signal that regulates epidermal lipid synthesis, which is associated first with the redeposition of stratum corneum lipids and then the normalization of stratum corneum barrier function.