We investigated the effects of the initial stiffness of a three-dimensional elastomer scaffold highly porous poly(glycerol sebacate)-on functional assembly of cardiomyocytes cultured with perfusion for 8 days. The polymer elasticity varied with the extent of polymer cross-links, resulting in three different stiffness groups, with compressive modulus of 2.35 +/- 0.03 (low), 5.28 +/- 0.36 (medium), and 5.99 +/- 0.40 (high) kPa. Laminin coating improved the efficiency of cell seeding (from 59 +/- 15 to 90 +/- 21%), resulting in markedly increased final cell density, construct contractility, and matrix deposition, likely because of enhanced cell interaction and spreading on scaffold surfaces. Compact tissue was formed in the low and medium stiffness groups, but not in the high stiffness group. In particular, the low stiffness group exhibited the greatest contraction amplitude in response to electric field pacing, and had the highest compressive modulus at the end of culture. A mathematical model was developed to establish a correlation between the contractile amplitude and the cell distribution within the scaffold. Taken together, our findings suggest that the contractile function of engineered cardiac constructs positively correlates with low compressive stiffness of the scaffold. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 26: 1382-1390, 2010