At present the involvement of cardiac valve interstitial cells (VICs) in growth, repair, and tissue engineering is understudied. Therefore, this study aims at characterizing ovine VICs in order to provide a solid base for tissue engineering of heart valves. Ovine ICs of the four heart valves were isolated by the explant outgrowth method and expanded in vitro up to passage 5. Vimentin and collagen I gene expression from freshly isolated or cultured ICs was measured by reverse transcriptase-polymerase chain reaction. Immunocytochemical stainings of vimentin, alpha-smooth muscle actin (ASMA), smooth muscle myosin, and procollagen I were performed on aortic VICs. In addition, migration and extracellular matrix deposition were studied in vitro in aortic VICs. ICs show stable vimentin and collagen I expression in culture. Expression is approximately doubled in cultured ICs compared with fresh isolates. More than 95% of ICs in each passage stain for vimentin and procollagen I. Freshly isolated ICs are ASMA and myosin negative, but ICs in culture partially stain for these contractile markers. ICs have stable matrix production for up to five passages, associated with stable migration of the cells. We conclude that ovine valve interstitial cells undergo phenotypic modulation to activated myofibroblasts under culture conditions but retain stable matrix production.