In this experimental study, we investigated the effect of dimethyl sulfoxide (DMSO) on the in vitro cytotoxicity of chemotherapeutic agents doxorubicin (Dox.) and mitomycin-C (Mito.-C), which are used in the intravesical chemotherapy for the treatment and prophylaxis of superficial bladder cancer. Materials and Methods: R4909 bladder cancer cell line (Science 176: 1337, 1972) was maintained as monolayer cultures in 75 $cm^2$ flasks in RPMI-1640 medium supplemented with %10 fetal calf serum (FCS). After trypsinization, $2xl0^5$ R4909 cells were distributed in sterile plates. On these prepared plates; DMSO, Mito.-C and Dox. was added at 1/2 dilutions separately and in combination with DMSO. After 72hours incubation, 3-(4,5-dimethylthiazol-2)2,5 diphenyl tetrozolium bromide (MTT) was added to the plates. These plates were centrifuged at 3000 rpm for 6 minutes to be read under 550 nm ELISA reader in order to obtain the optic density (OD) value which shows the cell survivaI. Results: According to OD values, DMSO had minimal effect on cell survival at concentrations equal and Iess than 4%. No enhancement of tumor cell kill by the addition of DMSO was observed. Conclusion: Our data indicate that, the addition of DMSO to drug solutions for intravesical chemotherapy at the time of treatment is unlikely to enhance the cytotoxic effect of the intravesical chemotherapeutic agents studied