In the absence of a metabolizing system (S9 mix) 4-chloro-o-toluidine (4-COT) was found to be ineffective in a combination of assays for gene mutations in Salmonella typhimurium, for chromosome aberrations and sister chromatide exchanges in human lymphocytes, and for the induction of spindle disturbances in V79 Chinese hamster cells. In the presence of S9, 4-COT was also ineffective in producing structural or numerical changes in mammalian cells, but the yields of 4-COT induced revertants in S. typhimurium strains TA 100 and TA 98 were about 2-fold higher than those in controls.