Hypertrophic scar (HTS) following thermal injury is a dermal fibroproliferative disorder that leads to considerable morbidity. The development of HTS involves numerous cell types and cytokines with dermal fibroblasts being a key cell. We have previously reported that the phenotype of fibroblasts isolated from HTS was altered compared to fibroblasts from normal skin. In this study, normal skin was horizontally sectioned into five layers using a dermatome from which fibroblasts were isolated and cultured. Cells from the deeper layers were observed to proliferate at a slow rate, but were morphologically larger. In ELISA and FACS assays, cells from the deeper layers produced more TGF-b1 and TGF-b1 producing cells were higher. In quantitative RT-PCR, the cells from the deeper layers had higher CTGF and HSP47 mRNA levels compared to those from superficial layers. In western blot, FACS and collagen gel assays, fibroblasts from the deeper layers produced more a-smooth muscle actin (a-SMA), had higher a-SMA positive cells and contracted collagen gels more. Fibroblasts from the deeper layers were also found to produce more collagen, but less collagenase by mass spectrometry and collagenase assay. Interestingly, cells from the deeper layers also produced more of the proteoglycan, versican, but less decorin. Taken together, these data strongly demonstrate that fibroblasts from the deeper layers of the dermis resemble HTS fibroblasts, suggesting that the deeper layer fibroblasts may be critical in the formation of HTS. Hypertrophic scarring is a common fibroproliferative disorder of the human dermis characterized by erythematous, raised, pruritic lesions of the healing skin, which is usually following thermal and other injuries that involve the deep dermis. 1 These lesions lead to scarring that compromises the appearance of healing skin and are commonly associated with contractures that limit movement and function of involved joints and facial features. As such, HTS is the principal factor that contributes to the prolonged and often uncomfortable rehabilitation period for thermally injured patients, particularly those who have survived large lifethreatening injuries, children, and individuals of dark-skinned races, in whom such scarring occurs more commonly. 2 The undesirable physical properties of HTS tissue can be attributed to the presence of a large amount of extracellular matrix that is of altered composition and organization, compared to normal dermis or mature scar. This matrix is the product of a dense population of fibroblasts, maintained in a hyperactive state by inflammatory cytokines such as TGF-b and other factors, some of which may be physical in origin. 3 Although the molecular and cellular events that lead to HTS have been extensively studied, the pathogenesis of this condition is still not well understood, making treatment difficult.Key cells involved in HTS are the dermal fibroblasts. Dermal fibroblasts are a dynamic and diverse population of cells whose function in skin and HTS in many respects remain unkn...