Skin graft contracture remains a significant cause of patient morbidity with reduction in joint mobility and cosmetic deformity. Despite recent advances, its mechanism is largely unknown. The authors have previously demonstrated the importance of the keratinocyte in the contraction of tissue-engineered skin in vitro. In this study, they investigate the effect of reducing keratinocyte differentiation on contraction by adding 0.8 mM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetra acetic acid (EGTA) to the culture medium and by culturing tissue-engineered skin submerged in medium rather than at air-liquid interface. They also simulate the effect of early mechanical splinting in vitro to study its effect on contraction. Here the study shows that removal of the epidermis after 16 days culture at air-liquid interface results in immediate dermal relaxation with a return to the original dermal surface area. Lowering extracellular calcium concentration with EGTA reduces keratinocyte differentiation and reduces the rate of contraction. Submerged culture does not significantly reduce differentiation of tissue-engineered skin and does not reduce contraction. However, following an initial short period of mechanical constraint, the rate of contraction of tissue-engineered skin is reduced. Reducing keratinocyte differentiation by lowering extracellular calcium with EGTA, reduces contraction. However, submerged culture of tissue-engineered skin is ineffective. A short period of splinting of meshed skin grafts during the initial phase of epithelialization and keratinocyte differentiation may be most effective in the prevention of subsequent contractures in vivo but additional studies are needed to establish this.