Cryopreserved valve homografts often fail in infants. Controversies are ongoing concerning the relative contributions of cryopreservation variables, immune responses, cellular viability, and durability of the extracellular matrix to the mode of tissue failure. In this study, cryosubstitution of conventionally cryopreserved heart valves, while still frozen, demonstrated extensive extracellular ice formation, with smaller crystals in the ventricularis than in either the spongiosa or fibrosa. Extracellular ice formation was prevented by vitrification, a process in which the biological system is stabilized as an amorphous solid in the absence of crystalline ice. It is proposed that the extensive ice formation observed in conventionally cryopreserved heart valves may cause extracellular matrix damage that predisposes the valves to calcification. Future studies will assess the above hypothesis by comparison of conventional and ice-free (vitrification) cryopreservation methods in animal models of calcification.