The relationship between extracellular Ca2+ concentration and EPSC amplitude was investigated at excitatory autapses on cultured hippocampal neurons. This relationship was steeply nonlinear, implicating the cooperative involvement of several Ca2+ ions in the release of each vesicle of transmitter. The cooperativity was estimated to be 3.1 using a power function fit and 3.3 using a Hill equation fit. However, simulations suggest that these values underestimate the true cooperativity. The role of different Ca2+ channel subtypes in shaping the Ca2+ dose-response relationship was studied using the selective Ca2+ channel blockers omega-agatoxin GIVA (omega-Aga), which blocks P/Q-type channels, and omega-conotoxin GVIA (omega-CTx), which blocks N-type channels. Both blockers broadened the dose-response relationship, and the Hill coefficient was reduced to 2.5 by omega-Aga and to 2.6 by omega-CTx. This broadening is consistent with a nonuniform distribution of Ca2+ channel subtypes across presynaptic terminals. The similar Hill coefficients in omega-Aga or omega-CTx suggest that there was no difference in the degree of cooperativity for transmitter release mediated via N- or P/Q-type Ca2+ channels. A model of the role of calcium in transmitter release is developed. It is based on a modified Dodge-Rahamimoff equation that includes a nonlinear relationship between extracellular and intracellular Ca2+ concentration, has a cooperativity of 4, and incorporates a nonuniform distribution of Ca2+ channel subtypes across presynaptic terminals. The model predictions are consistent with all of the results reported in this study.