Many hybridoma and recombinant myeloma cell lines have been successfully adapted to growth in protein-free media. Compared with serum-supplemented media, use of protein-free media promotes superior cell growth and protein expression and facilitates downstream purification of the expressed product. Owing to its sterol auxotrophy, the NS0 myeloma is normally grown in either a serum-supplemented medium or a serum-free medium supplemented with an animal-derived lipoprotein. CD Hybridoma Medium (a protein-free, chemically defined formulation) grows many cell lines that do not exhibit lipid dependence, but this medium does not support growth of NS0 cells without further lipid supplementation. We tested several commercially available lipid supplements in CD Hybridoma Medium, including bovine EX-CYTE VLE. None of the tested supplements supported long-term growth of NS0 cells in CD Hybridoma Medium. Sustained long-term growth of NS0 cells was achieved in CD Hybridoma Medium supplemented with various animal- or plant-derived lipids complexed with cyclodextrin. NS0 cells adapted to CD Hybridoma Medium supplemented with cyclodextrin-lipid complex reached peak cell densities that were more than double those observed in serum-supplemented medium and were cultured for more than 15 passages. These cultures were also successfully cryopreserved and recovered in this defined medium. Through the use of cyclodextrin-based additives to CD Hybridoma Medium, it is possible to solubilize significant quantities of sterols and other lipids and to maintain a protein-free, chemically defined cultivation environment for NS0 cells. The culture system can be kept entirely free of animal-derived components if the supplement is made with plant-derived or synthetic lipids.