It has been reported that human keratinocyte cell line (HaCaT cells) are a valuable tool for studying the metabolism of chemicals that penetrate the skin. However, little is known about the genotoxicity and carcinogenicityin HaCaT cellson skin cancer study of carcinogens, such as inorganic arsenic.Therefore, the present studyinvestigated genotoxicity in HaCaT cells by the in vitro micronucleus (vitMN) test. The vitMN test is a method that typically uses cultured human or rodent cells. It provides a comprehensive basis for investigating chromosome damaging potential in vitro because both aneugens and clastogens can be detected. Mitomycin C (MMC; a DNA cross linking agent) and Colchicine (COL; an aneugen) were used as positive controls. One of the most important considerations in the performance of the vitMNtest is ensuring that the cells being scored have completed mitosis during the treatment or the post-treatment incubation period, if one is used. Therefore,following exposure to MMC and COL were harvested after an additional 1.5 to 2.0 times the normal cell cycles. This study was composed of two parts; with cytochalasinB(cyto B) and without cyto B.Positive controls should be used at concentrations expected to give reproducible and detectable increase over negative control which demonstrated the sensitivity of vitMN test. The present study investigated to define the appropriate test concentrations of two positive controlsat significantly increases number of MN in the range of 55±5% cytotoxicity of HaCaT cells. As a result, the appropriate MMC concentrations for short treatment with cyto B and without cyto B were 60 and 10μg/ml, respectively. But 30 μg/ml was selected at COL concentration for short treatment, regardless of cyto B. For continuous treatment, respective 4 and 0.25 μg/ml were as appropriate MMC concentration, and respective 0.2 and 0.1 μg/mlwere as appropriate COL concentrations with cyto B and without cyto B.To the best of our knowledge, this is the first study to establishfor genotoxicity onHaCaT cells in association with carcinogenesis. The present study will provide an important insight into the genotoxicity on HaCaT cells with respect to the genotoxic chemicals.