Ito cells are the primary cell type in the liver responsible for increased type I collagen production observed during fibrogenesis. After a fibrogenic stimulus, Ito cells change from their normal quiescent state to an activated state. In this study, we examined the expression of the α1(I) collagen gene in Ito cells that were freshly isolated from normal rat liver (quiescent) and Ito cells that were activated by culture on plastic. Northern blots showed that α1(I) collagen messenger RNA levels were low in freshly isolated Ito cells and Ito cells cultured on plastic for up to 4 days, but were induced after 7 days in culture. To determine the location of important transcriptional regulatory elements within the 5′-flanking region of the α1(I) collagen gene, transient transfections using various portions of the α1(I) collagen gene promoter linked to the luciferase gene were performed in cultured Ito cells. As in fibroblast cells, only 220 base pairs (bp) of immediate 5′-flanking sequence of the collagen α1(I) gene was necessary for efficient expression of the reporter gene. Deoxyribonuclease I footprinting analysis and mobility shift assays showed dramatic differences in the DNA binding proteins that interact with the 220 base pair promoter region of the α1(I) collagen gene between freshly isolated and activated Ito cells. Sp1 binding activity was greatly increased in activated Ito cells, as was binding activity for CCAAT binding factor (CBF), a transcription factor previously shown to interact with the α2(I) collagen gene promoter. The increased Sp1 binding activity to the α1(I) collagen gene promoter involves a posttranscriptional event, as shown by Northern and Western blots. Therefore, we show that activated Ito cells have a different pattern of transcription factor binding activity interacting with the α1(I) collagen gene promoter compared with quiescent Ito cells, including increased Sp1 binding activity, and that this may contribute to the increase in α1(I) collagen gene expression during fibrosis. (HEPATOLOGY 1995; 22:241–251.)