In vitro skeletal muscle engineering involves the culture of isolated primary myogenic cells in an environment conducive to the formation of a three-dimensional (3D) tissue construct capable of generating force. Isolated human myogenic cells have been used to study cell–cell interactions, permitting identification of functions intrinsic to skeletal muscle in two dimensions (2D). However, the independent contribution of human myogenic and non-myogenic cell types that comprise skeletal muscle to myogenic cell differentiation, force generation and matrix remodelling has yet to be established in 3D. The objective of this study was to use isolated human myogenic and non-myogenic muscle-derived cells (MDC) seeded in 3D collagen constructs to engineer a biomimetic craniofacial skeletal construct. The aim was to purify the two subpopulations of myogenic and non-myogenic cells from human masseter muscle and quantitate myogenic cell differentiation, force generation and matrix remodelling of the 3D collagen construct. The results showed that both the heterogeneous mixture of cells and the purified myogenic cell population expressed myogenin, indicative of myogenic cell differentiation. Further, there was a synergistic effect as the heterogeneous co-culture of myogenic and non-myogenic cells generated the highest peak force and expressed the most MMP-2 mRNA compared to isolated individual cell populations. Copyright © 2008 John Wiley & Sons, Ltd.