We have examined the MGH-U1 human bladder carcinoma cell line and 12 primary bladder carcinoma biopsies for their ability to form spheroids in suspension culture and in multiwell dishes. MGH-U1 cells formed tightly packed spheroids with a necrotic center and viable rim whereas three sublines formed loose aggregates only. Spheroids formed from as few as 100 MGH-U1 cells placed into multiwells. MGH-U1 cells derived from spheroids formed new spheroids more rapidly and and consistently than cells derived from monolayer culture. Spheroid diameter increased at a rapid rate of approximately 100 microns/d in multiwell dishes, and necrosis occurred only in spheroids of diameter greater than 1 mm. Spheroids placed in spinner culture at a higher concentration (approximately 1.5 spheroids/ml) grew more slowly and developed necrosis at smaller diameters. The width of the viable rim of spheroids grown in spinner culture was maintained at approximately 190 microns over a wide range of spheroid diameters (400 to 1000 microns). Sequential trypsinization of spheroids, which stripped layers of cells from the spheroids, demonstrated no difference in the plating efficiency of cells derived from varying depths into the spheroid. Only one of the 12 primary bladder biopsy specimens demonstrated an ability to form spheroids. This biopsy, designated HB-10, formed spheroids that grew linearly over 40 d, formed colonies in methylcellulose culture and grew as xenografts in immune-deprived mice. These studies characterize the MGH-U1 spheroids that are useful in vitro models to study the effects of various treatments for solid tumors and demonstrate the limited capacity of cells from primary human bladder biopsies to form spheroids.