We compared the cytotoxicity of vincristine in MGH-U1 human bladder cancer cells growing as exponential monolayer culture, spheroids and xenografts. Cells treated as spheroids were resistant to vincristine as determined by clonogenic survival and growth delay. The spheroid population had a smaller proportion of cells in G2 + M than monolayer cells. Cell derived from increasing depths of the spheroid viable rim had similar cell cycle distribution characteristics and sensitivity to vincristine. Prolonged treatment of spheroid did not increase vincristine cytotoxicity significantly. When cells derived from spheroids were treated as monolayers, the cytotoxicity was the same as that of cells maintained as monolayer cultures. The vincristine resistance observed in spheroids was also observed in xenografted tumours treated in vivo. Vincristine decreased the clonogenic survival of xenografted cells at in vivo doses which were greater than the LD10 for the mice. The in vitro cytotoxicity of the xenografted tumours at these lethal doses was similar to that of cells treated as spheroids. We conclude that vincristine resistance in spheroids may be attributed in part to the small proportion of cells traversing mitosis but not to the development of intrinsic resistance by passage through spheroid growth. Our results are consistent with cell cycle kinetics and limited penetration contributing to vincristine resistance in spheroids. The spheroid system can serve as a model of in vivo cytotoxicity for antineoplastic agents with cell cycle phase specificity such as vincristine.