1. A technique has been developed to monitor neurotransmitter release simultaneously with intracellular Ca2+ concentration ([Ca2+]i) in single release boutons whose diameters range from 3 to 5 microns. 2. Using this technique, we have found a highly non-linear relationship between the rate of asynchronous release and [Ca2+]i. The Hill coefficient lies between 3 and 4. 3. The affinity (Kd) of the putative release-related Ca2+ receptor for asynchronous release was calculated to be in the range of 2-4 microM. 4. The same range of values of Hill coefficient and Kd were obtained when [Ca2+]i was elevated both by bath application of ionomycin and by repetitive stimulation at high frequency. 5. Our results show that the Ca2+ receptor(s) associated with asynchronous release exhibits high affinity for Ca2+.