The hepatic vitamin A-storing Ito cell has been implicated as a causative cell in hepatic fibrogenesis. Using a modification of a recent method (Friedman, S. L., Roll, F. J., Boyles, J., and Bissell, D. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8681-8685), rat Ito cells were isolated and passaged in vitro on collagen-coated plastic dishes through cell generation 40-50. The collagen synthetic phenotype for Ito cells grown on various extracellular matrices was demonstrated by immunofluorescence and quantitated by competition enzyme-linked immunosorbent assays. When grown on a type I collagen matrix, Ito cells produced type IV greater than type III greater than type I collagen. When grown on a type IV collagen matrix, the cells produced relatively equal amounts of types I and III collagen. The absolute amounts of type I collagen produced were greater when cells were grown on type IV versus type I matrix. When 10(-5) M retinol was added to cell cultures, there was a uniform increase in type III collagen regardless of matrix type but a decrease in type I collagen when cells were grown on a type IV matrix and a large increase in type I collagen when cells were grown on a type I collagen matrix. The levels of cellular retinol binding protein, a key cytosolic retinol transport protein, were quantitated by high performance liquid chromatography and compared for cells grown on type I versus type IV collagen matrices. It was found that cells on a type I matrix contain 4.96 +/- 2.8 times more cellular retinol binding protein than do cells grown on a type IV matrix. In conclusion, Ito cell collagen synthesis may be altered by underlying extracellular matrix and exogenous retinol. This in vitro culture system should allow the study of regulatory factors and possible therapeutic anti-fibrogenic mediators.