Diastolic dysfunction is characterized by prolonged relaxation, increased filling pressure, decreased contraction velocity, and reduced cardiac output. Phenotypical features of diastolic dysfunction can be observed at the level of the isolated myocyte. This article reviews the cellular mechanisms that control relaxation at the level of the myocyte in the healthy situation and discusses the alterations that can affect physiologic function during disease. It focuses specifically on the mechanisms that regulate intracellular calcium handling, and the response of the myofilaments to calcium, including the changes in these components that can contribute to diastolic dysfunction.