The proliferative activity of Ito cells in acute liver injury induced by carbon tetrachloride (CCl4) was studied by light and electron microscopic autoradiography. At 48 hours after a single intraperitoneal injection of CCl4, the livers of the mice given vitamin A per os for preceding 9 days and those of the mice without vitamin A-pretreatment were removed. Small tissue blocks of each group were respectively incubated at 37 degrees C for 1 hour in culture medium containing 3H-thymidine. After CCl4 injection, perisinusoidal and sinusoidal cells adjacent to centrilobular necrotic liver cells increased in number and size. Some of them were labelled by 3H-thymidine. On the other hand, the perisinusoidal and sinusoidal cells in the peripheral zone in which liver cells are not markedly degenerated nor necrotic showed no noticeable increase in number. They contained very few or no silver grains after 3H-thymidine. In control mice the labelling of perisinusoidal cells was hardly observed. Electron microscopic autoradiography revealed that most of the labelled perisinusoidal cells in the centrilobular zone possess characteristics of Ito cells in their location and in the fine structures such as the presence of small fat droplets, well-developed rough endoplasmic reticulum, and Golgi complex in the cytoplasm. These findings indicate that Ito cells incorporate 3H-thymidine in DNA synthesis after hepatocellular necrosis resulting in cell proliferation.