We previously devised a magnetic resonance imaging method that allows for the nondestructive and quantitative determination of glycosaminoglycan concentration in excised cartilage. The technique measures the concentration of the charged contrast agent Gd-DTPA2− (gadolinium diethylenetriaminepentaacetic acid) equilibrated within cartilage, from which the tissue distribution of glycosaminoglycan can be calculated. The goals of our study were to determine the practicality of nondestructively monitoring glycosaminoglycan concentration in cartilage explants over a long-term culture period and to determine if glycosaminoglycan could be restored to glycosaminoglycan-depleted cartilage explants maintained in long-term culture. To meet our objectives, we harvested bovine cartilage explants, treated them initially with trypsin to reduce the glycosaminoglycan concentration, and cultured them for as long as 8 weeks. Images depicting glycosaminoglycan concentration were calculated from magnetic resonance images acquired at selected intervals during the trypsinization process and the subsequent culture period. The results indicate that gadolinium-enhanced magnetic resonance imaging can follow the reduction of glycosaminoglycan concentration over the course of enzymatic digestion and the replenishment of glycosaminoglycan over several weeks of culture and that cultured cartilage explants are capable of restoring glycosaminoglycan to 85% of its initial concentration. Of particular interest, samples cultured for 5 weeks indicated a depth dependence of glycosaminoglycan regeneration to values similar to the initial physiologic distribution. Thus, this magnetic resonance imaging method may be a very powerful means for exploring the spatial and temporal evolution of glycosaminoglycan in cartilage.