Cell suspensions derived from 2 specimens of normal human urothelium and 13 human transitional cell carcinomas were studied by discontinuous density gradient centrifugation. The histochemical and proliferative properties of the fractions were evaluated. In normal urothelium, DNA synthesis was restricted to a subpopulation of small round cells which could be partly separated from the more numerous pyramidal and giant cells on the basis of their higher physical density. Normal urothelium did not form colonies in agar. In well-differentiated tumors, the majority of cells present were low-density, elongated cells which histochemically resembled the differentiated pyramidal cells of normal tissue, but DNA synthesis and the ability to form colonies in agar were restricted to a subpopulation of high-density small round cells. Colony size analysis showed that intermediate-density bands contain cells capable of a few divisions which do not form large colonies. With increasing tumor grade, there was an apparent shift into the high-density clonogenic compartment.