An understanding of the mechanical and mechano-molecular responses that occur during the differentiation of mouse c2c12 myoblasts in 3-D culture is critical for understanding growth, which is important for progress towards producing a tissue-engineered muscle construct. We have established the main differences in force generation between skeletal myoblasts, dermal fibroblasts, and smooth muscle cells in a 3-D culture model in which cells contract a collagen gel construct. This model was developed to provide a reproducible 3-D muscle organoid in which differences in force generation could be measured, as the skeletal myoblasts fused to form myotubes within a collagen gel. Maintenance of the 3-D culture under sustained uni-axial tension, was found to promote fusion of myoblasts to form aligned multi-nucleate myotubes. Gene expression of both Insulin Like Growth Factor (IGF-1 Ea) and an isoform of IGF-1 Ea, Mechano-growth factor (IGF-1 Eb, also termed MGF), was monitored in this differentiating collagen construct over the time course of fusion and maturation (0–7 days). This identified a transient surge in both IGF-1 Ea and MGF expression on day 3 of the developing construct. This peak of IGF-1 Ea and MGF expression, just prior to differentiation, was consistent with the idea that IGF-1 Ea stimulates differentiation through a Myogenin pathway [Florini et al., 1991: Mol. Endocrinol. 5:718–724]. MGF gene expression was increased 77-fold on day 3, compared to a 36-fold increase with IGF-1 Ea on day 3. This indicates an important role for MGF in either differentiation or, more likely, a response to mechanical or tensional cues. Cell Motil. Cytoskeleton 54:226–236, 2003. © 2003 Wiley-Liss, Inc.