Hyaluronan is an extracellular matrix component implicated in expansion of the extracellular space, organization of supramolecular architecture, cell motility, proliferation, tumour metastases and wound healing. Hyaluronan is highly expressed in the developing heart but it is only a minor component of the mature heart. The loss of hyaluronan synthase-2 (Has2) results in embryonic lethality with a phenotype remarkably similar to that of the versican-deficient heart defect mouse. Has2-deficient embryos lack hyaluronan-containing cardiac jelly, and at embryonic day 9.5 show arrested development, with an apparent absence of the right ventricle and underdevelopment of the conustruncus segment, and pericardial effusion consistent with heart failure. Cardiac cushions are totally absent, and endocardial cell migration over collagen gels is not detectable in Has2-deficient atrioventricular (AV) canal explants. Endothelial to mesenchymal transformation is also defective in AV explants from Has2-null embryos. The normal phenotype is restored in AV canal explants from Has2-deficient embryos by co-culture with wild type AV canal explants, with conditioned media from wild type AV explants or with exogenous hyaluronan. These results provide evidence for a direct role for hyaluronan during endocardial cushion and AV canal morphogenesis.