Changes in the intracellular concentration of free ionized calcium ([Ca(2+)]i) control a host of cellular processes as varied as vision, muscle contraction, neuronal signal transmission, proliferation, apoptosis etc. The disturbance in Ca(2+)-signaling causes many severe diseases. To understand the mechanisms underlying the control by calcium and how disorder of this regulation relates to pathological conditions, it is necessary to measure [Ca(2+)]i. The Ca(2+)-regulated photoproteins which are responsible for bioluminescence of marine coelenterates have been successfully used for this purpose over the years. Here we report the results on comparative characterization of bioluminescence properties of aequorin from Aequorea victoria, obelin from Obelia longissima, and clytin from Clytia gregaria charged by native coelenterazine and coelenterazine analogues f, i, and hcp. The comparison of specific bioluminescence activity, stability, emission spectra, stopped-flow kinetics, sensitivity to calcium, and effect of physiological concentrations of Mg(2+) establishes obelin-hcp as an excellent semisynthetic photoprotein to keep track of fast intracellular Ca(2+) transients. The rate of rise of its light signal on a sudden change of [Ca(2+)] is almost 3- and 11-fold higher than those of obelin and aequorin with native coelenterazine, respectively, and 20 times higher than that of the corresponding aequorin-hcp. In addition, obelin-hcp preserves a high specific bioluminescence activity and displays higher Ca(2+)-sensitivity as compared to obelin charged by native coelenterazine and sensitivity to Ca(2+) comparable with those of aequorin-f and aequorin-hcp.