Hepatic sinusoidal fat-storing Ito cells are felt to represent the primary storage site for hepatic vitamin A and may be important collagen-producing effector cells during hepatic fibrogenesis. The cirrhotic liver generally has a decreased vitamin A content with increased numbers of “transitional” myofibroblasts adjacent to developing fibrous bands. It has been suggested that Ito cells “transform” into these myofibroblasts. The in vivo loss of Ito cell vitamin A can be simulated in vitro as Ito cells spontaneously lose their vitamin A lipid droplets during primary culture. The current study evaluated Ito cell proliferation in vitro with respect to vitamin A content and the extracellular collagen matrix. The cells were grown on a Type I or Type IV collagen matrix to simulate the types of collagens presumed to be present in the space of Disse. Initially it was observed that freshly isolated Ito cells begin to proliferate several days after isolation coincident with the decline of the vitamin A lipid droplets and a decrease in cellular retinyl palmitate. The proliferation rate for passaged Ito cells was similar on either matrix (on Type I collagen: T1/2 = 2.2 ± 1.1 days, n = 16; on Type IV collagen: T1/2 = 3.3 ± 1.4 days, n = 4; p < 0.11). This proliferation rate remained constant through Cell Generation 16 and was similar to the rate for primary Ito cells in culture. To evaluate the possibility that primary Ito cell proliferation is causally related to the loss of vitamin A, Ito cells were re-exposed to an increased concentration of retinol in vitro. Passaged Ito cells (grown on a Type I or Type IV collagen matrix) which lack vitamin A droplets were exposed to media containing 10−5M retinol. Cell proliferation was markedly inhibited for the 2 weeks of observation without apparent loss of cell viability. The effect was noted to be reversible and dose related with a submaximal effect at 10−6M retinol. In conclusion, retinol can modulate Ito cell proliferation in vitro and may represent an important controlling factor with respect to Ito cell behavior.