The calcium titration experiments were performed with 9 uM and 100 uM concentrations of Arsenazo III (Az) at various pH°s in the presence as well as in the absence of ethylene glycol-bis-(2-amino ethylether)-N,N°-tetra-acetic acid (EGTA). Analysis of results indicated that in the presence of EGTA in 9 uM Az solutions the Ca-Az stoichiometry changes from 1:1 to two Ca binding with one Az (2:1) upon increase in pH from 6.55 to 7.26. In the same systems but without EGTA the binding stoichiometry remains 1:1 throughout the pH range 6.55 to 7.32. In case of 100 uM Az III, however, EGTA has no effect on Ca-Az stoichiometry and it is 1:1 in the above pH range. An estimate of dissociation constants (KD) in EGTA containing systems showed a simultaneous decrease in KDby three orders of magnitude with a change in stoichiometry from 1:1 to 2:1 in the case of 9 uM Az III. At 100 uM dye concentration, with no change in 1:1 binding stoichiometry, KDalso remained constant throughout the pH range studied. We suggest that at lower dye concentration a competition between Ca-Az and Ca-EGTA binding causes this change in stoichiometry. The discrepancy among the previous reports about Ca-Az stoichiometry appeared to be due to change in pH in the presence of EGTA.