Abstract Introduction: A major challenge associated with heart valve tissue engineering is the in vitro creation of mature tissue structures compliant with native valve functionality. Various cell types have been investigated for heart valve tissue engineering. In addition to prenatal, umbilical cord- and vascular-derived cells, mesenchymal stem cells (MSCs) have gained large interest for tissue engineering purposes, because of their broad differentiation potential. However, bone marrow derived MSCs require a highly invasive harvesting procedure and decline in both cell number and differentiation potential proportionally with the donor’s age. In contrast, adipose derived stem cells (ADSCs) represent an interesting alternative. The ease of repeated access to subcutaneous adipose tissue as well as the less invasive donation procedures provide clear advantages. Therefore, this study investigated the suitability of ADSCs as alternative cell source for tissue engineered heart valves (TEHVs). Methods: Human ADSCs were seeded on TEHV-scaffolds (n=11) made of nonwoven polyglycolic acid coated with poly-4-hydroxybutyrate. TEHVs were cultivated in diastolic-pulse-duplicator-bioreactor systems and subsequently seeded with a superficial layer of ADSC-derived endothelial cells. Quantitative assessment of extracellular matrix composition of the TEHV-leaflets was performed with biochemical analyses for sulphated glycosaminoglycans, hydroxyproline and DNA content. Microstructural evaluation was performed on representative samples of the TEHVleaflets by (immuno-)histochemistry and scanning electron microscopy. The mechanical properties of the ADSC derived TEHV-leaflets were characterized by biaxial tensile tests. Results: ADSC-derived TEHV-leaflets showed a homogenous vital cell distribution throughout the whole leaflet structure that consisted of large amounts of glycosaminoglycans and collagen and was endothelialized. Furthermore, the mechanically stable matrix of the ADSC-derived TEHVs showed a stiffness range in the right order of magnitude for heart valve applications. Conclusion: Human ADSCs represent a promising alternative autologous mesenchymal cell source for TEHVs that is of large clinical relevance due to their easy accessibility, efficient proliferation and excellent tissue formation capacities.