A bacterial endotoxin test (BET) is required to detect or quantify bacterial endotoxin that may be present in radiopharmaceutical preparations. The test uses Limulus amebocyte lysate, which, in the presence of bacterial endotoxin and divalent calcium ions, causes the formation of a coagulin gel. 99m Tc-labeled radiopharmaceuticals have chelating ligands such as diethylene triamine pentaacetic acid (DTPA), ethylene dicysteine (EC), L,L-ethyl cysteinate dimer (ECD), N- [2,4,6-trimethyl-3 bromoacetanilid] iminodiacetic acid (mebrofenin), dimercapto succinic acid-III (DMSA-III), dimercapto succinic acid-V (DMSA-V), and several others, which form a coordination complex with Na-99m Tc-O 4 in the presence of reducing agents. During BET by the gel-clot method, the free sulfhydryl (-SH) and carboxyl (-COOH) in some of the chelating agents in the final 99m Tclabeled radiopharmaceuticals decrease the free divalent calcium ion concentration, which in turn inhibits coagulin gel formation. This study was designed using the premise that addition of calcium chloride solution to the reaction mixture would nullify this effect. Methods: We present here the data obtained from BET assay analysis of 99m Tc-labeled radiopharmaceuticals and the cold kits from which they are made (EC, ECD, methoxyisobutylisonitrile, DTPA, mebrofenin, methylene diphosphonic acid [MDP], DMSA-III, and DMSA-V) using 2 different dilutions, maximum valid dilution (MVD) and half maximum valid dilution (MVD/2), with and without the addition of calcium chloride at a final concentration of 300 μM. Results: It was observed that at MVD and MVD/2 all of the 99m Tc-labeled kits exhibited interference in coagulin gel formation with the exception of 99m Tcmethoxyisobutylisonitrile, 99m Tc-MDP, 99m Tc-mebrofenin, and 99m Tc-ECD. However, only the cold kits of methoxyisobutylisonitrile and MDP did not show inhibition. An addition of calcium chloride solution nullified this interference at both MVD and MVD/2 in all of the 99m Tclabeled radiopharmaceuticals in which interference was observed. Conclusion: In practice, Limulus amoebocyte lysate testing is not a method of choice for 99m Tc-labeled radiopharmaceuticals because these radiopharmaceuticals exhibit interference. However, our study proves the hypothesis that the addition of calcium chloride can circumvent this problem. The addition of calcium chloride provides an enhanced biologic quality control testing option for the final formulation of 99m Tc-labeled radiopharmaceuticals at the hospital radiopharmacy end. In the centralized radiopharmacy laboratories of hospitals, different generator-based radiopharmaceuticals such as 99m 68 Ga-, and 90 Y-labeled molecules are being used for various diagnostic and therapeutic purposes. 99m Tclabeled radiopharmaceuticals have wider applications in SPECT and planar imaging with g cameras because of their short half-life (6.03 h) and pure g emission (140 keV). It is mandatory for hospital radiopharmacies to monitor the bacterial endotoxin levels of a 99m Tc-labeled radiopharmac...