Cell surface phosphatidylethanolamine (PE) of the yeast cell was probed by biotinylated Ro09-0198 (Bio-Ro), which specifically binds to PE and was visualized with fluorescein-labelled streptavidin. In Saccharomyces cerevisiae, the signals were observed at the presumptive bud site, the emerging small bud cortex, the bud neck of the late mitotic large-budded cells and the tip of the mating projection. In Schizosaccharomyces pombe, the signals were observed at one end or both ends of mono-nucleated cells and the division plane of the late mitotic cells. These sites were polarized ends in the yeast cells, implying that PE is exposed on the cell surface at cellular polarized ends. Treatment of S. cerevisiae cells with Ro09-0198 resulted in aberrant F-actin accumulation at the above sites, implying that limited surface exposure of PE is involved in the polarized organization of the actin cytoskeleton. Furthermore, S. cerevisiae ros3, dnf1 and dnf2 null mutants, which were known to be defective in the internalization of fluorescence-labelled PE, as well as the combinatorial mutants, were stained with Bio-Ro at the enlarging bud cortex, in addition to the Bio-Ro-staining sites of wild-type cells, suggesting that Ros3p, Dnf1p and Dnf2p are involved in the retrieval of exposed PE at the bud cortex.