Physiologic evidence suggests that local activation of the cerebellar granule cell layer produces a much more restricted spatial activation of overlying Purkinje cells than would be expected from the parallel fiber system. These results have led to the suggestion that synapses associated with the ascending granule cell axon may provide a large, direct, excitatory input to Purkinje cells, whereas parallel fiber synapses may be more modulatory in nature. In the current experiments, serial electron microscopy was used to reconstruct synapses associated with these two segments of the granule cell axons in the cerebellar cortex of albino rats. The results indicate that there are significantly more presynaptic vesicles in ascending segment synapses than in parallel fiber synapses. Furthermore, a first-order linear regression analysis revealed positive correlations between all measures of pre- and postsynaptic morphology for parallel fibers, but not for ascending segment synapses. Perhaps most surprisingly, serial reconstructions of postsynaptic spines and their associated dendrites demonstrated that spines contacted by ascending segment synapses are located exclusively on the smallest diameter distal regions of the Purkinje cell dendrites, whereas parallel fiber synapses are found exclusively on intermediate- and large-diameter regions of the spiny branchlets. Based on two independent calculations, we estimate that 20% of the granule cell synapses onto a Purkinje cell are actually made by the ascending segment. By using computer simulations of a single Purkinje cell dendrite, we have also demonstrated that synchronous activation of these distal ascending segment inputs could produce a substantial somatic response. Taken together, these results suggest that the two different regions of granule cell axons may play very different physiologic roles in cerebellar cortex.