The effects of photodynamic therapy (PDT) alone and in combination with Mitomycin C (MMC) on WiDr cells, a human colon adenocarcinoma cell line, were investigated. The addition of MMC increased the cytotoxicity of PDT. The presence of MMC resulted in a reduction or a removal of the shoulder of the PDT survival curves as well as an increase in their slopes. Increasing with the concentrations of MMC from 0.01 to 0.025 micrograms/ml, the cytotoxic effects of the two treatments changed from additivity to supra-additivity as judged by comparing dose-response curves for each treatment alone with survival curves after combination therapy and by isobologram analysis. The cytotoxicity of MMC could also be enhanced by a practically nontoxic treatment of PDT (8% cell inactivation). The cytotoxicities of MMC and PDT in combination were found to be dependent on the sequence of the two treatments. When MMC (> or = 0.02 micrograms/ml) and Photofrin II were given simultaneously for 16 h and then followed by irradiation, the combination was found to be more effective than when MMC was given to the cells immediately after PDT and kept in the medium for 16 h. Possible mechanisms of the combination effects of PDT and MMC are discussed briefly.