Cultured skin substitutes (CSS) consisting of autologous fibroblasts and keratinocytes combined with biopolymers are an adjunctive treatment for large excised burns. CSS containing two cell types are limited by anatomical deficiencies, including lack of a vascular plexus, leading to slower vascularization after grafting than split-thickness autograft. To address this limitation, CSS were prepared containing human keratinocytes, fibroblasts, and dermal microvascular endothelial cells (HDMEC) isolated from a single skin sample. After 16 days in culture, control CSS and CSS containing HDMEC (CSS+EC) were grafted to full-thickness wounds in athymic mice. In CSS+EC in vitro, HDMEC persisted in the dermal substitutes and formed multicellular aggregates. One wk after grafting, HDMEC in CSS+EC organized into multicellular structures, some containing lumens. By 4 wk after grafting, HDMEC were found in linear and circular organizations resembling vascular analogs associated with basement membrane deposition. In some cases, colocalization of HDMEC with mouse perivascular cells was observed. The results demonstrate HDMEC transplantation in a clinically relevant cultured skin model, persistence of HDMEC after grafting, and HDMEC organization into vascular analogs in vitro and in vivo. All cells were derived from the same donor tissue, indicating the feasibility of preparing CSS containing autologous HDMEC for grafting to patients.—Supp, D. M., Wilson-Landy, K., Boyce, S. T. Human dermal microvascular endothelial cells form vascular analogs in cultured skin substitutes after grafting to athymic mice.