Many sea urchins, including the strongylocentroid Hemicentrotus pulcherrimus, produce an amniotic cavity on the left for adult rudiment formation at the late larval stage. In contrast, temnopleurids form a cell mass at the early larval stage instead of an amniotic cavity. Although the mechanisms establishing left-right polarity of the amniotic cavity involve cell-cell interactions and signaling pathways, corresponding pathways for the cell mass are unknown. We analyzed the effects of blastomere isolation on the specification of larval axes in the temnopleurid Temnopleurus toreumaticus and compared them to those in H. pulcherrimus. Blastomere isolation at the two- or four-cell stages in T. toreumaticus disturbed the location of the cell mass and adult rudiment in approximately 10-20% of specimens. In contrast, isolation at the two-cell stage in H. pulcherrimus caused the left-right polarity to become random. When blastomeres isolated at the two-cell stage were cultured as pairs, approximately 20% of pairs had atypical polarity in both species. Following isolation at the four-cell stage, 71.4% of quartets produced larvae with atypical polarity in T. toreumaticus. Thus, cell-cell interaction between two daughter blastomeres after the second cleavage may be involved in the mechanism determining left-right polarity. Dye injection into a blastomere and subsequent observations indicated that the location of the boundary of the first cleavage showed similar patterns in both species. These observations suggest that species-specific mechanisms establish the larval axes and blastomeres at the two- and four-cell stages redistribute their cytoplasm, forming gradients that establish left-right polarity.