Nestin, an intermediate filament protein, is expressed in the neural stem cells of the developing central nervous system. This tissue-specific expression is driven by the neural stem cell-specific enhancer in the second intron of the nestin gene. In this study, we showed that the mouse nestin gene was expressed in pluripotent embryonic carcinoma (EC) P19 and F9 cells, not in the differentiated cell types. This cell type-specific expression was conferred by the enhancer in the second intron. Mutation of the conserved POU factor-binding site in the enhancer abolished the reporter gene expression in EC cells. Oct4, a Class V POU factor, was found to be coexpressed with nestin in EC cells. Electrophoretic mobility-shift assays and supershift assays showed that a unique protein-DNA complex was formed specifically with nuclear extracts of EC cells, and Oct4 protein was included. Together, these results suggest the functional relevance between the conserved POU factor-binding site and the expression of the nestin gene in pluripotent EC cells.