Green S was fed to rats of both sexes, over three generations, at dietary concentrations designed to provide daily intakes of 0, 50, 500 or 1000 mg Green S/kg body weight. At each generation, treated groups each consisted of 36 males and 36 females with 60 of each sex as controls. The F0 generation first received Green S as weanlings, but succeeding generations were exposed throughout life, including in utero, as treatment continued during gestation and lactation. There were no adverse effects of treatment on body-weight gain, food and water consumption or on the general condition of the animals. Green-coloured faeces were produced by all animals exposed to the colouring and pale green coloration of urine or the bladder was seen in a few animals at autopsy. The post-mortem examinations and organ weights of animals receiving up to 500 mg Green S/kg/day showed no adverse effects of treatment. At 1000 mg/kg/day findings related to treatment were increased spleen weight (both sexes) and increased kidney weight (male), but relevant histopathological changes were not seen in either of these organs. Caecal enlargement was the most consistent finding in animals receiving 500 or 1000 mg Green S/kg/day, but this was not considered to be a toxic effect. Reproductive performance and intra-uterine development were not affected by treatment despite green colouring being visible in the amniotic sacs of foetuses from dams given 500 or 1000 mg Green S/kg/day. Small differences in the degree of skeletal ossification of foetuses from F2 generation dams were not related to treatment. A slightly premature eruption of the incisors during postnatal development of treated animals was not considered to be an adverse effect. It is concluded that the no-untoward-effect level in this study is 500 mg Green S/kg body weight/day.