CALCIFICATION IN ECHINODERMS: EFFECTS OF TEMPERATURE AND DIAMOX ON INCORPORATION OF CALCIUM-45 IN VITRO BY REGENERATING SPINES OF STRONGYLOCENTROTUS PURPURATUS. 1. Calcification during regeneration of experimentally fractured spines of the sea urchin, Strongylocentrotus purpuratus (Stimpson), was studied quantitatively under different conditions with calcium-45 as a tracer. 2. Fractured spines rapidly incorporated 45Ca in vivo or in vitro after a lag period of about two days. The lag period is attributed to wound healing and reorganization of tissue at the site of fracture. 3. Additional experiments were conducted while calcification was in progress by allowing fractured spines to regenerate for four days in vivo followed by incubation in 45Ca in vivo or in vitro up to 24 hours. In these experiments incorporation of the label was nearly linear with time and no significant difference was observed in the rate of uptake of 45Ca between regenerating spines incubated in vivo and those from the same urchin incubated simultaneously in vitro. 4. Incorporation of 45Ca in vitro was directly proportional to temperature between 4.7° and 20° C, at which a maximum occurred. A temperature of 26° C appeared to be lethal and little incorporation of 45Ca took place. Values of Q10 and the energy of activation varied inversely with temperature, with overall means of 2.72 and 15,504 calories per mole, respectively, between 4.7° and 20° C. 5. Diamox (acetazolamide) at concentrations from 10-3 to 10-6 M, inhibited incorporation of 45Ca in vitro by 50% to 61%. It is inferred from these results that carbonic anhydrase is involved in calcification of regenerating spines of S. purpuratus.