LIBRA is a fluorescent biosensor of inositol 1,4,5-trisphosphate (IP(3)) and is composed of the ligand-binding domain of the rat type 3 IP(3) receptor and cyan and yellow fluorescent proteins. We examined the responses of LIBRA and its IP(3)-insensitive mutant LIBRA-N to compounds known to inhibit IP(3)-induced Ca(2+) release. Heparin, a competitive antagonist of IP(3) receptors, increased the emission ratio of LIBRA but not that of LIBRA-N. In contrast, 2-aminoethoxydiphenyl borate, a known non-competitive inhibitor of IP(3) receptor, decreased the emission ratios of both LIBRA and LIBRA-N. Thus, the concurrent use of LIBRA-N with LIBRA identifies nonspecific responses. These results indicate that LIBRA and its mutant control can be used to detect specific agonists and antagonists of IP(3) receptors. We also demonstrate the utility of LIBRA and LIBRA-N in discriminating between specific and nonspecific responses in intact cells.