Cell‐level mechanical and 3D spatial cues are essential to the organization and architecture of new tissues that form during growth, repair or in bioreactors. Fibroblast‐seeded 3D collagen constructs have been used as bioartifical extracellular matrix (ECM) providing a 3D environment to embedded resident cells. As cells attach to scaffold fibrils, they generate quantifiable contractile forces which depend on cell type, cell attachment, cell density, growth factors, and matrix stiffness. The aim of this study was to quantify the cytomechanical and molecular responses of human dermal (HDF) and neonatal foreskin fibroblasts (HNFF) seeded in constructs of increased stiffness. We also tested the effect of blocking early attachment using serum starvation on these outputs. Constructs were placed under uniaxial strains of 0–10% to increase scaffold stiffness, prior to gel contraction, and force generation was monitored using a tensional culture force monitor (t‐CFM). Increased matrix stiffness reduced generation of quantifiable cellular force (up to 70%) over 24 h in both cell types and delayed the onset of measurable contraction (upto sevenfold). The delay of measurable force generation was cell lineage dependent but not FCS dependent. Gene expression of MMP‐2, TIMP‐2, and collagen type III expression in HDFs were significantly upregulated in constructs of increased stiffness. HNFFs did not show any significant changes in these gene expressions indicating a lineage specific response. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2007