Abstract The formation of calcified spicules in cultures of micromeres isolated at the 16-cell stage from eggs of the sea-urchin, Hemicentrotus pulcherrimus, was inhibited by ethacrynic acid and furosemide, which are inhibitors of chloride transport, as well as by nifedipine, a Ca 2+ antagonist. 14CO 2 generated by cultured cells exposed to [U- 14C]glucose was incorporated into spicules. The 14CO 2, produced in the cells was regarded as being an intracellular CO 2 source. The production of 14CO 2, was only slightly inhibited by the compounds studied. The release of intracellular 14CO 2 from the cells was inhibited by ethacrynic acid and furosemide, but was not blocked by nifedipine. Cl -/HCO - 3 ATPase activity, which is sensitive to chloride transport inhibitors, was also detected in the precipitate fraction obtained by centrifuging the homogenate of micromere-derived cells (30 h of culture at 20° C) at 105,000 g for 60 min. The incorporation of intracellular 14CO 2 as well as of 45Ca 2+ into the spicules was strongly inhibited by the three compounds. The inhibition of 14CO 2 incorporation into CaCO 3, in spicules by nifedipine was probably due to the shortage of Ca 2+ resulting from the blockage of Ca 2+ channels. It seems likely that HCO - 3 release from primary mesenchyme cells is necessary for the calcification of spicules and is coupled with chloride transport across the cell membrane resulting from the reaction with Cl -/HCO 3 --ATPase, which is sensitive to these chloride transport inhibitors.