Adult mesenchymal stem cells (MSCs) are a promising cell source in tissue engineering due to their availability, ease of isolation and high proliferative activity. This study was undertaken to investigate whether immortalised human MSC are able to undergo chondrogenic differentiation when cultured in alginate or in resorbable scaffolds. We directly compared chondrogenesis MSCs with that of human nasoseptal chondrocytes. Two previously established human stem cell lines L87/4 and V54-2 immortalised using the SV40 large T-antigen were either cultured in alginate or in polyglycolic acid/poly-L-lactic acid (PGA/PLLA) (90/10) copolymer scaffolds. TGF-β1 was added for induction of chondrogenesis. Human nasoseptal chondrocytes and human fibroblasts were used as controls. Cultures were analysed for sulfated glycosaminoglycans (alcian blue staining) and for the presence of collagen type I, II and X (immunolabelling). SV40 large T-antigen immortalised human MSCs have the potential to undergo chondrogenic differentiation: After 21 days, cartilage-specific type II collagen was present in alginate and PGA/PLLA scaffolds, independent of the addition of TGF-β1. Collagen type X was present in monolayer cultures as well as in alginate and PGA/PLLA scaffolds. Collagen type I was produced in marginal amounts only. Immortalised human MSCs are a suitable tool to study chondrogenesis in vitro and to screen biomaterials for cartilage tissue engineering applications.