1. The kinetics and stoicheiometry of the Ca2+-activated luminescent reaction of the photoprotein obelin were studied at different temperatures and in the presence of various substances, including the physiologically occurring cations K+, Na+, Ca+, Mg2+ and H+. 2. The results suggest Ca2+-independent rates of rise and fall in obelin luminescence following sudden changes in [Ca2+] and indicate that changes in [Ca2+] over the range 1 x 10(-6) - 3 x 10(-4) M are followed significantly faster by the obelin response (approx. 3 ms delay at 20 degrees C) than by the aequorin response (approx. 10 ms delay at 20 degrees C). 3. Obelin was found to emit low-intensity light (less than 10(-6) of the maximum Ca2+-activated response), which was independent of Ca2+ at concentrations below about 10(-7) M. The level of this Ca2+-independent light emission is sensitive to temperature and the ionic composition of the solution. 4. The log-log plot of light intensity against ionized Ca indicates a maximum slope of 2.5, suggesting the involvement of three Ca ions in the luminescent reaction. 5. Increase in the concentration of K+, Na+, Mg2+ and H+ generally shift the Ca2+ activation curve for obelin toward higher Ca2+ concentrations. These cations can also affect the maximum rate of obelin utilization at more extreme concentrations. 6. The maximal rate of obelin utilization was also affected to varying degrees by the presence of uncharged substances such as glucose, sucrose and polyvinylpyrrolidone. However, neither the sensitivity of obelin to Ca2+ nor the quantum yield were modified by the substances. 7. Caffeine (less than 20 mM), procaine (less than 20 mM) and sodium dantrolene (saturated solution), substances known to modify cellular Ca2+ movements, had little effect on the Ca2+-induced luminescent reaction. The general anaesthetics chlorpromazine and halothane appeared to lower greatly the quantum yield without, however, modifying the maximum rate of obelin utilization. 8. A scheme of reaction for obelin activation by Ca2+ is presented which adequately explains the experimental observations and allows one to make accurate predictions regarding the relative obelin response under a variety of ionic conditions at room temperature.