Human bladder tumor fragments were cultured on collagen gel. In this system, the three dimensional architecture, cell-to-stroma and cell-to-cell interactions, and tumor heterogeneity were maintained. Cell viability and labeling index (LI) were determined by exposure to 3H-thymidine and autoradiography. Of the samples from 20 patients with transitional cell carcinoma, 14 (70%) were successfully cultured and had a mean LI of 32%. In addition, one specimen from a patient with squamous cell carcinoma was cultured and had a LI of 61%. Cultured samples were tested for chemosensitivity using a two hour exposure of mitomycin C in concentrations ranging from one to 50 micrograms./ml. A dose-dependent relationship was demonstrated; LI decreased as mitomycin C concentrations increased. The methodology described provides an alternative to suspension or monolayer techniques of culturing human bladder tumors for pharmacological studies.