Degeneration of the nucleus pulposus (NP) has been implicated as a major cause of low back pain. Tissue engineering strategies may provide a viable NP replacement therapy; however, culture conditions must be optimized to promote functional tissue development. In this study, a standard serum-containing medium formulation was compared to a chemically defined, serum-free medium to determine the effect on matrix elaboration and functional properties of NP cell-laden carboxymethylcellulose (CMC) hydrogels. Additionally, both media were further supplemented with transforming growth factor-beta 3 (TGF-β3). Glycosaminoglycan (GAG) content increased in both TGF-β3-treated groups and was highest for treated, serum-free constructs (9.46 ± 1.51 µg GAG/mg wet weight), while there were no quantifiable GAGs in untreated serum-containing samples. Histology revealed uniform, interterritorial staining for chondroitin sulfate proteoglycan throughout the treated, serum-free constructs. Type II collagen content was greater in both serum-free groups and highest in treated, serum-free constructs. The equilibrium Young's modulus was highest in serum-free samples supplemented with TGF-β3 (18.54 ± 1.92 kPa), and the equilibrium weight swelling ratio of these constructs approached that of the native NP tissue (22.19 ± 0.46 vs. 19.94 ± 3.09, respectively). Taken together, these results demonstrate enhanced functional matrix development by NP cells when cultured in CMC hydrogels maintained in serum-free, TGF-β3 supplemented medium, indicating the importance of medium formulation in NP construct development. Biotechnol. Bioeng. 2010; 105: 384–395. © 2009 Wiley Periodicals, Inc.