A tri-copolymer sponge consisting of gelatin, hyaluronic acid, and chondroitin sulfate (GHC) was designed to mimic the cartilage environment in vivo for cartilage regeneration. The present study aimed to temporally characterize the magnetic resonance relaxation time of GHC constructs in vivo in a rodent heterotopic model. GHC sponges with cells (GHCc) or without cells (GHC) implanted in rat leg muscle were monitored using MRI (4.7 T MR scanner) on day 0, 7, 14, and 21 after implantation. The results revealed that the transverse relaxation time (T2) of GHC constructs decreased significantly over time when compared to the T2 of GHCc constructs. However, the longitudinal relaxation time (T1) of GHCc and GHC constructs remained stable. Moreover, hematoxylin and eosin and immunohistochemical staining with antibodies to S100 protein, and types I and II collagen showed that normal morphology, phenotype, and function of chondrocytes were preserved in the GHCc construct. Thus, we concluded that GHC constructs adequately support chondrocyte growth and function. On top of that, T2 may be a useful tool for monitoring cartilage regeneration in GHC constructs. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.