Purpose The development of multidrug resistance is a problem in chemotherapy for many tumors. In vitro models of multidrug resistance require adapted cell strains that are conventionally produced from parental lines by chronic low dose drug exposure. Because adjunctive intravesical chemotherapy for superficial bladder cancer uses short courses of high dose treatment, we investigated whether such exposure of the RT112 cell line (Catalogue No. ACC 418, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) to mitomycin C, which is a common intravesical agent, would elicit multidrug resistance. Materials and Methods Three 1-hour exposures to graded concentrations were done at 3-week intervals. The highest mitomycin C concentrations permitting recovery in cultures and, therefore, available for examination were 3.13 and 1.06 μg/ml. Cross-resistance to epirubicin in surviving cultures was visualized by confocal microscopy and quantified by MTT residual viable biomass assay. Spheroids were made by the agarose technique and exposed to high dose mitomycin C to assess the probability that the relevant concentrations might be found clinically in some cell layers of a superficial lesion. Results Resistance was induced by 3 short drug exposures. The evidence for this was functional (MTT assay) and by intracellular localization. Toxicity to an alternative multidrug resistance class drug was lowered in surviving clones and nuclear exclusion of the drug was noted. Spheroid experiments showed sharp gradients of incorporated drug across the outermost layers of cells, suggesting that a proportion of cells in clinical superficial bladder cancer would be exposed to drug at concentrations that generated the resistant clones in these experiments. Conclusions We report multidrug resistance induction using 2 independent methodologies. The results have implications for the development of experimental models and the likelihood of resistance resulting from clinical regimens. Brief exposure can elicit detectable resistance. It is arguable that selective rather than instructive mechanisms are involved, and the levels of drug required are likely to exist in a superficial transitional cell carcinoma frond exposed at its surface to high drug concentrations.