2-Mercaptobenzimidazole (MBI) and its methyl derivatives 4-methyl-MBI (4-MeMBI), 5-methyl-MBI (5-MeMBI), and 4(or 5)-methyl-MBI (4(5)-MeMBI) are widely applied industrial agents with substantial thyrotoxicity and hepatotoxicity detected in rats in vivo. Here, we examined the in vitro cytotoxicity of MBI and its derivates in cultured SIRC rabbit corneal cells. SIRC cells were cultured in the presence of the test chemicals for 72 hr, and cell viability was determined by estimating the number of cells using a crystal violet staining assay. The median lethal concentration (LC₅₀) was calculated for each of the chemicals. The methyl derivatives showed higher cytotoxicity than MBI, which is in contrast to previous in vivo findings demonstrating higher thyrotoxicity and hepatotoxicity of MBI compared to its derivates. According to the LC₅₀ values, the ranking of the tested agents in terms of cytotoxicity was 5-MeMBI (761.5 µM) ≥ 4-MeMBI (796.3 µM) ≥ 4(5)-MeMBI (822.9 µM) > MBI (1002.9 µM). The present results suggest that the lower thyrotoxicity and hepatotoxicity of methyl derivatives of MBI is related to their faster detoxification in vivo, because SIRC cells are considered to have lower drug-metabolizing activity than hepatic cells.