To study the Ca2+ dependency of luteinizing hormone-releasing hormone (LHRH) release in the bullfrog sympathetic ganglia, a method was developed to fill the preganglionic nerve terminal boutons with membrane-impermeant fura-2. We found that as stimulation frequency increased from 0.5 to 40 Hz, the peak [Ca2+]i ([Ca2+]p) and the rate of rise in [Ca2+]i increased, the decay of [Ca2+]i transients followed up to three exponentials, and release of LHRH was linearly related to integral of ([Ca2+]i--[Ca2+]t)dt. The threshold level of [Ca2+]i for LHRH release for a given set of boutons on a C cell, [Ca2+]t, was estimated by the [Ca2+]p evoked by 0.5 Hz stimulation that does not induce LHRH release.