In many neurons, subthreshold somatic depolarization can spread electrotonically into the axon and modulate subsequent spike-evoked transmission. Although release probability is regulated by intracellular Ca(2+), the Ca(2+) dependence of this modulatory mechanism has been debated. Using paired recordings from synaptically connected molecular layer interneurons (MLIs) of the rat cerebellum, we observed Ca(2+)-mediated strengthening of release following brief subthreshold depolarization of the soma. Two-photon microscopy revealed that, at the axon, somatic depolarization evoked Ca(2+) influx through voltage-sensitive Ca(2+) channels and facilitated spike-evoked Ca(2+) entry. Exogenous Ca(2+) buffering diminished these Ca(2+) transients and eliminated the strengthening of release. Axonal Ca(2+) entry elicited by subthreshold somatic depolarization also triggered asynchronous transmission that may deplete vesicle availability and thereby temper release strengthening. In this cerebellar circuit, activity-dependent presynaptic plasticity depends on Ca(2+) elevations resulting from both sub- and suprathreshold electrical activity initiated at the soma.