Human heart valve interstitial cells (ICs) have been understudied to date. The aim of this study was to compare the phenotype and reactivity of ICs cultured from valves, pericardium and skin. Immunofluorescence studies showed that 57% of ICs from valves and 21% of ICs from pericardium, but less than 0.5% of skin fibroblasts, expressed smooth muscle a -actin. Intracellular calcium changes evaluated by loading cells with fura-2 acetoxymethyl ester demonstrated that vasoactive agents induced transient increases in intracellular calcium: skin fibroblasts were least responsive. Heart valve ICs are a mixed population of specific cell types, many of which express SM a -actin and may be classified as myofibroblasts. Intracellular calcium responses to vasoactive agents indicated that a number of receptor signaling pathways existed. Evaluation of these may elucidate the role of myofibroblasts and other fibroblast phenotypes in valve function/dysfunction, as well as contribute to the development of tissue-engineered valves.