Abstract 4-Chloro- o-phenylenediamine (4-C- o-PDA) is a liver carcinogen in mice and was found to be weakly mutagenic in the liver of female Big Blue™ mice after short term treatment. In the present study the test compound was given subchronically in the diet for 26 weeks at doses of 0, 5000 and 10,000 ppm. The corresponding average test substance intake was 2166 mg kg −1 day −1 (males: 1794 mg kg −1 day −1; females: 2539 mg kg −1 day −1) and 4610 mg kg −1 day −1 (males: 3926 mg kg −1 day −1; females 5925 mg kg −1 day −1) at the low and high dose, respectively. After sacrifice, tissues were flash frozen in liquid nitrogen. The lacI mutant frequency in the liver was determined from three male and three female mice per dose group. The genomically integrated transgene was recovered by packaging into λ phage using Transpack™ packaging extract (Stratagene, La Jolla, USA) followed by infection of Escherichia coli strain SCS-8. Blue mutant plaques were scored against a background of clear non-mutant plaques. Food consumption decreased initially at 10,000 ppm, while no treatment related effect on food intake was observed at 5000 ppm. Body weight gain was found to be decreased in all treated animals. Absolute and relative liver weight increased in a dose-related manner, but only the latter effect was statistically significant. A clear dose dependent increase in lacI mutant frequencies was observed in the liver of both sexes. The following mutant frequencies (×10 −5) were observed: 2.73±1.01 (males, untreated), 7.24±1.50 (females, untreated), 18.91±5.30 (5000 ppm, males), 24.91±7.58 (5000 ppm, females), 20.47±6.68 (10,000 ppm, males) and 36.17±14.98 (10,000 ppm, females). It is therefore concluded that 4-C- o-PDA is a strong mutagen in the liver of mice treated subchronically for 26 weeks.