Cardiac looping, which begins with ventral bending and rightward rotation of the primitive heart tube, is an essential morphogenetic event that occurs early in vertebrate development. The biophysical mechanism that drives this process is unknown. It has been speculated that increased stiffness along the dorsal side of the ventricle combined with an intrinsic cardiac force causes the heart to bend. There is no experimental support for this hypothesis, however, since little is known about regional mechanical properties of the heart during looping. We directly measured diastolic stiffness of the inner curvature (IC), outer curvature (OC), and dorsal-ventral sides of the stage 12 chick heart by microindentation. The IC of intact hearts was found to be significantly stiffer than either the OC or the sides. which were of similar stiffness. Isolated cardiac jelly, which is a thick, extracellular matrix compartment underlying the myocardium, was approximately an order of magnitude softer than intact hearts. The results of a computational model simulating the indentation experiments, combined with the stiffness measurements, suggests the regional variation in stiffness is due to the material properties of the myocardium. A second model shows that a relatively stiff IC may facilitate bending of the heart tube during looping.