Abstract Objective The repair of osteochondral defects with chondrocytes genetically modified to express desired growth factors promises great potential in orthopaedic therapy. Controlled expression of the transgenes is required in many instances. The objective of the present study was to demonstrate the inducibility of tetracycline-responsive transgene expression in osteochondral defects in the knee joint filled with genetically modified chondrocyte implants. Methods An expression plasmid containing the lacZ gene under the control of the minimal CMV promoter fused to the Tet-responsible element (TRE) as well as the reverse transactivator (rtTA2 s -M2) was constructed and used to transfect isolated articular chondrocytes from New Zealand white rabbits. rtTA2 s -M2 binds to the TRE in the presence of tetracycline and leads to the transcription of the transgene. Different concentrations of DNA and various DNA:lipid ratios were tested to determine best transfection conditions. Transfection efficiency and inducibility were analysed by histochemical analysis and flow-cytometry. To evaluate the system in vivo, collagen-sponges were seeded with transfected autologous chondrocytes and implanted in osteochondral defects in the knees of NZW-rabbits. Gene expression was induced by doxycycline and 3 weeks later, LacZ-expression in isolated knee joints was evaluated in histological sections by X-gal staining. Results In vitro 13.5% (±1.32) of induced primary chondrocytes were LacZ-positive, while non-induced controls showed a background-staining in 0.6% (±0.2). In vivo, upon doxycycline treatment, induction of lacZ-gene-expression could be demonstrated in chondrocytes in 3-week-old, well-integrated implants. Conclusion For the first time, tetracycline-inducible gene expression is demonstrated to work in the treatment of osteochondral defects. This demonstrates the feasibility for a gene therapy-assisted approach using controlled expression of therapeutic growth factors from transplanted genetically modified chondrocytes.