Direct administration of therapeutic candidate gene sequences using the safe and effective recombinant adeno-associated virus (rAAV) vectors is a promising strategy to stimulate the biologic activities of articular chondrocytes as an adapted tool to treat human osteoarthritic (OA) cartilage. In the present study, we developed a combined gene transfer approach based on the co-delivery of the pleiotropic transformation growth factor beta (TGF-β) with the specific transcription factor SOX9 via rAAV to human normal and OA chondrocytes in vitro and cartilage explants in situ in light of the mitogenic and pro-anabolic properties of these factors. Effective, durable co-overexpression of TGF-β and SOX9 significantly enhanced the levels of cell proliferation both in human normal and OA chondrocytes and cartilage explants over an extended period of time (21 days), while stimulating the biosynthesis of key matrix components (proteoglycans, type-II collagen) compared with control conditions (reporter lacZ gene transfer, absence of vector treatment). Of further note, expression of hypertrophic type-X collagen significantly decreased following co-treatment by the candidate vectors. The present findings show the value of combining the transfer and expression of potent candidate factors in human OA cartilage as a means to re-establish essential features of normal cartilage and counteract the pathological shift of homeostasis. These observations support the concept of developing dual therapeutic rAAV gene transfer strategies as future, adapted tools for the direct treatment of human OA. This article is protected by copyright. All rights reserved.