BACKGROUND: The method to generate bioengineered skin constructs was pioneered several decades ago, and nowadays these constructs are used regularly for the treatment of severe burns and non-healing wounds. Commonly, these constructs are comprised of skin fibroblasts within a collagen scaffold, forming the skin dermis, and stratified keratinocytes overlying this, forming the skin epidermis. In the past decade there has been a surge of interest in bioengineered skins, with researchers searching for alternative cell sources, or scaffolds, from which constructs can be established, and for more biomimetic equivalents with skin appendages. OBJECTIVES: In this manuscript we wanted to evaluate whether human hair follicle dermal cells can act as an alternative cell source for engineering the dermal component of engineered skin constructs. METHODS: We established in vitro skin constructs by incorporating into the collagenous dermal compartment either primary interfollicular dermal fibroblasts, hair follicle dermal papilla, or hair follicle dermal sheath cells. In vivo skins were established by mixing dermal cells and keratinocytes in chambers on top of immunologically compromised mice. RESULTS: All fibroblast subtypes were capable of supporting growth of overlying epithelial cells, both in vitro and in vivo. However, we found hair follicle dermal sheath cells to be superior to fibroblasts in their capacity to influence the establishment of a basal lamina. CONCLUSIONS: Human hair follicle dermal cells can be readily interchanged with interfollicular fibroblasts, and used as an alternative cell source for establishing the dermal component of engineered skin both in vitro and in vivo. This article is protected by copyright. All rights reserved.