Metabolism of free and esterified cholesterol and triacylglycerol was compared in cultured neuronal cells, glial cells, and fibroblasts grown from chick embryos. Cellular contents of free and esterified cholesterol were comparable in these cells and triacylglycerol content in the neuronal cells was about 40% of that in the other cell types. Cholesterol synthesis from [3H]acetate was high in all these cells and was not affected by fetal calf serum in the culture medium. Monensin, which has been shown to influence cholesterol metabolism through the inhibition of low-density lipoprotein receptor recycling in human fibroblasts, did not induce profound effects on cholesterol metabolism in these cells. Higher incorporation of [3H] oleic acid into esterified cholesterol was observed in the glial cells and fibroblasts when fetal calf serum was removed from the culture medium. Cellular content of the esterified cholesterol also increased in the glial cells under a serum-free arrangement. 25-Hydroxycholesterol induced higher incorporation of both [3H]acetic acid and [3H]oleic acid into esterified cholesterol in all of these cells. The results indicate that the active metabolism of cholesterol found in cultured chick neural cells and fibroblasts may not be regulated by an LDL receptor-mediated system and some factors in fetal calf serum inhibit cellular accumulation of esterified cholesterol.