The present study was performed to clarify the effects of a 4-min exposure of mitomycin C (MMC) on cell growth, the cell cycle and MMC dose incorporated into DNA, using Chang’s cultured human conjunctival cells. A low dose of MMC ranging from 0.00025 to 0.004% showed dose-dependent cytotoxicity when cell growth was active. Fifty percent cell viability was found when cells were treated with 0.001% MMC. A flow cytometer showed that 0.001% MMC inhibited the DNA synthetic phase. After 0.04% MMC was exposed to 3 ×10⁶ cells and immediately rinsed, DNA was isolated to measure the dose of MMC detected from DNA. The total amount of DNA was 7 µg from which 3 µg of MMC was detected by high performance liquid chromatography. The above results revealed that the lowest concentration of MMC which caused 50% cell viability and cell cycle inhibition was 0.001% and that MMC was rapidly incorporated into DNA.