Primary cultures and cell lines were established from suspensions of purified fat‐storing cells isolated from the rat liver. When seeded at a suitable density, fat‐storing cells in primary culture reached confluency in 3 to 4 days and could be transferred and established as cell lines for at least two passages. The typical morphological characteristics of fat‐storing cells in vivo were retained in the cells during primary culture. Vitamin A fluorescence was still associated with lipid droplets of cells in culture up to and including the second passage. Investigation of the cytoskeletal structure by indirect immunofluorescence showed the presence of vimentin, actin and tubulin in the cells; no a‐prekeratin was present. The presence of vimentin suggested a fibroblastic or possible myogenic origin for fat‐storing cells. The presence of connective tissue components in fat‐storing cells in culture was demonstrated by indirect immunofluorescence. Collagen Types I and IV and laminin were present intracellular in small granules in fat‐storing cells in primary culture and in the first passage. Cells in the fourth passage contained only collagen Type 1. Fibronectin was only aligned extracellularly along the cell membrane, which did not exclude an extracellular source. Rat liver fat‐storing cells in culture show a high proliferating capacity. Cell multiplication during prolonged culture was associated with phenotypic transition to a more fibroblastic appearance and gradual disappearance of vitamin A. These results indicate that fat‐storing cells may be among the cell types involved in pathological changes observed during development of liver fibrosis.