The degree of conservation among phyla of early mechanisms that pattern the left-right (LR) axis is poorly understood. Larvae of sea urchins exhibit consistently oriented LR asymmetry. The main part of the adult rudiment is formed from the left coelomic sac of larvae, the left hydrocoel. Although this left preference is conserved among all echinoderm larvae, its mechanism is largely not understood. Using two marker genes, HpNot and HpFoxFQ-like, which are asymmetrically expressed during larval development of the sea urchin Hemicentrotus pulcherrimus, we examined in this study the possibility that the recently discovered ion flux mechanism controls asymmetry in sea urchins as it does in several vertebrate species. Several ion-transporter inhibitors were screened for the ability to alter the expression of the asymmetric marker genes. Blockers of the H(+)/K(+)-ATPase (omeprazole, lansoprazole and SCH28080), as well as a calcium ionophore (A23187), significantly altered the normal sidedness of asymmetric gene expression. Exposure to omeprazole disrupted the consistent asymmetry of adult rudiment formation in larvae. Immuno-detection revealed that H(+)/K(+)-ATPase-like antigens in sea urchin embryos were present through blastula stage and exhibited a striking asymmetry being present in a single blastomere in 32-cell embryos. These results suggest that, as in vertebrates, endogenous spatially-regulated early transport of H(+) and/or K(+), and also of Ca(2+), functions in the establishment of LR asymmetry in sea urchin development.