The stellate cells of the liver are the main storage site of retinyl esters in the body. During cultivation in vitro of stellate cells isolated from rat and rabbit livers were observed that the cells rapidly loose their retinyl ester content. Freshly isolated stellate cells contain about 144 nmol of total retinol/mg of protein, while cells cultivated for 14 days contained below 0.1 nmol/mg of protein. When 3-day-old cultures were incubated for 6 h with 2 microM retinol, the cellular content increased from 5.6 to approx. 9.4 nmol of total retinyl esters/mg of protein. In contrast, little retinyl ester accumulated in 10-20-day-old cultures incubated with 2 microM retinol. At 50 microM retinol, however, the retinyl ester level did increase both with 3-day-old cultures and 10-20-day-old cultures. In parallel experiments with cultured fibroblasts esterification characteristics similar to those seen in older cultures of stellate cells were observed. When 10-day-old cultures of stellate cells were incubated with retinol alone, or in combination with palmitic acid, linoleic acid or oleic acid, the total storage of retinyl esters increased by 20-150%. In most cases, the fatty acid supplemented in the medium was found to be the dominant fatty acid esterified with retinol. Cultures of stellate cells were then exposed to a physiological concentration (1.3 microM) of radioactive retinol free in solution or bound to retinol-binding protein. With 3-day-old cultures, as well as older cultures, the cellular content of unesterified retinol was 10-20 times higher when free retinol was added compared with addition of retinol bound to retinol-binding protein. However, 2-3-fold as much radioactive retinyl esters were recovered in cells incubated with retinol-retinol-binding protein compared with retinol free in solution. These results show that retinol delivered to stellate cells from retinol-binding protein is preferentially esterified, and that the complex is handled differently to free retinol by the stellate cells.