The gel formation by endotoxin with limulus amoebocyte lysate (LAL) solution requires the presence of free calcium ions. The chelating agents and radiopharmaceuticals prepared from the chelating agents always reduce the available free calcium levels present in the kits used for the testing of apyrogenicity and thus inhibit gel formation of LAL with E. Coli endotoxin (ET). This inhibition of gel formation could be reversed by the addition of excess free calcium ions or the excessive dilution of radiopharmaceuticals and chelating agents. The tests of positive control (0.2 ml ET units and LAL), inhibition control (0.1 ml sample, 0.1 ml ET and LAL), and negative control (0.1 ml sterile saline and LAL) were carried out with the fresh preparation (0.1 ml) of samples (triplicate), tropolone, ACD anticoagulant, and 99mTc-labeled radiopharmaceuticals. In the Ca-supplemented tests, 0.1 ml of 60 mM sterile calcium chloride solution was added to the test solutions and incubated for 60 min at 37 degrees centigrade. The results of gel formation (+ve and -ve) and normalization of inhibition control tests with Ca-supplement indicate that commercial LAL kits need extra calcium ions for the correct testing of the apyrogenicity of chelate-containing radiopharmaceuticals and chelating agents.