Vitamin A (retinol) and its active derivatives (retinoic acids) are essential for growth and development of the mammalian fetus. Maternally derived retinol must pass the placenta to reach the developing fetus. Despite its apparent importance, little is known concerning placental transfer and metabolism of retinol, and particularly of placental production and storage of retinyl esters. To elucidate this metabolic pathway, we incubated, in the presence of retinol, 1) human full-term placental explants and 2) primary cultures of major cells types contributing to placental function: trophoblasts and villous mesenchymal fibroblasts. We used HPLC to determine the types and concentrations of retinyl esters produced by these explants and cells. About 14% of total cellular retinol in placental explants was esterified. The most abundant esters were myristate and palmitate. Primary cell cultures showed that fibroblasts efficiently produced retinyl esters, but trophoblasts did not. In both types of experiments, no retinyl esters were detected in the culture medium, suggesting that retinyl esters were produced for storage purpose. These results suggest that villous mesenchymal fibroblasts are primary sites of retinol esterification and storage in the placenta.