Recent studies have suggested that transmitter release facilitation at synapses is largely mediated by presynaptic Ca2+ current facilitation, but the exact contribution of Ca2+ current facilitation has not been determined quantitatively. Here, we determine the contribution of Ca2+ current facilitation, and of an increase in the residual free Ca2+ concentration ([Ca2+](i)) in the nerve terminal, to paired-pulse facilitation of transmitter release at the calyx of Held. Under conditions of low release probability imposed by brief presynaptic voltage-clamp steps, transmitter release facilitation at short interstimulus intervals (4 ms) was 227 +/- 31% of control, Ca2+ current facilitation was 113 +/- 4% of control, and the peak residual [Ca2+](i) was 252 +/- 18 nm over baseline. By inferring the 'local' [Ca2+](i) transients that drive transmitter release during these voltage-clamp stimuli with the help of a kinetic release model, we estimate that Ca2+ current facilitation contributes to similar to 40% to paired-pulse facilitation of transmitter release. The remaining component of facilitation strongly depends on the build-up, and on the decay of the residual free [Ca2+](i), but cannot be explained by linear summation of the residual free [Ca2+](i), and the back-calculated 'local' [Ca2+](i) signal, which only accounts for similar to 10% of the total release facilitation. Further voltage-clamp experiments designed to compensate for Ca2+ current facilitation demonstrated that about half of the observed transmitter release facilitation remains in the absence of Ca2+ current facilitation. Our results indicate that paired-pulse facilitation of transmitter release at the calyx of Held is driven by at least two distinct mechanisms: Ca2+ current facilitation, and a mechanism independent of Ca2+ current facilitation that closely tracks the time course of residual free [Ca2+](i).