Tumor spheroids were cultured from five human glioma cell lines which differed considerably in their relative amount and composition of glycosaminoglycans (GAG), flbronectin and other extracellular matrix (ECM) components when grown as mololayer cultures. These differences were also evident when the cells were grown as spheroids. Under the 3-dimensional geometry of the spheroid system, there was, however, generally a more extensive ECM. Especially noteworthy was the presence of a small proteoglycan, probably a dermatan sulphate proteoglycan, in the ECM of the spheroids, but not in the monolayers. Noteworthy was also the appearance of flbronectin in spheroids which did not show any staining for flbronectin when grown as monolayer. The two spheroid types (U-87MG, U-105MG) with the most extensive matrix, and with the lowest proportion of hyalucronic acid (HA), had a low proliferation rate, whereas the three other spheroid types (U-118MG, U-138MG, U-251MG) with a less extensive ECM, and a relatively high production of HA had a much higher proliferation rate. These data provide further evidence for the usefulness of culturing cell lines as spheroids in the process of understanding important cell biological phenomena.