Pluripotent, feeder-dependent teratocarcinoma cell lines were cultured without a feeder layer in a medium containing 10(-4) M beta-mercaptoethanol (beta-medium) and compared for the development of early markers with cells cultured with a feeder layer. The cells cultured in beta-medium lost the PNA (peanut agglutinin) receptor typically at low density. This change was accompanied with enhanced secretion of plasminogen activator and the loss of sensitivity to anti-F9 serum, indicating the stem cell differentiation. In contrast, the cells cultured on a feeder layer did not show any marker changes, thus indicating the lack of differentiation. These results indicate that while the presence of feeder cells inhibits cell differentiation, cultivation in beta-medium permits the differentiation of pluripotent teratocarcinoma cells.