Repair of cartilage defects involves sequential participation of specific hormones and growth factors with potential impairment by inflammatory cytokines. We explored an in vivo gene therapy treatment to supply adenoviral vectors carrying the genes of interleukin-1 receptor antagonist protein (IL-1ra) and insulinlike growth factor-1 (IGF-1), hoping to enhance repair of full-thickness equine chondral defects treated with microfracture. We asked whether our treatment could (1) increase proteoglycan and Type II collagen content in the repair tissue, (2) improve the macroscopic and histomorphometric aspect of the repair tissue, and (3) induce prolonged and increased IL-1ra and IGF-1 production in treated joints. Twelve horses had full-thickness chondral defects created in their carpus and stifle followed by microfracture. Joints were injected with either equine IL-1ra/IGF-1 adenoviral preparation or Gey's balanced salt solution. Sixteen weeks later, defect healing was evaluated macroscopically, histologically, histochemically, and biochemically. Production of IL-1ra and IGF-1 was measured by enzyme-linked immunosorbent assay and radioimmunoassay. We found increased proteoglycan content in treated defects along with augmented Type II collagen associated with substantial transgene expression of IL-1ra during the first 3 weeks. These data suggest in vivo gene therapy can improve biologic processes associated with chondral defect repair.