Aspartate (Asp) and/or glutamate (Glu) have been proposed as putative excitatory transmitters released from synaptic terminals of the olivo-cerebellar climbing fiber afferents to the Purkinje cells. Investigations of the climbing fiber transmitter(s) separately for hemispheres and vermis were performed to examine whether the current controversy over the role of Asp as a neurotransmitter in the climbing fibers may be due to topographic differences. K(+)-induced Ca2(+)-dependent release of endogenous substances was investigated in slices of cerebellar hemisphere and vermis of control rats and those deprived of climbing fibers by 3-acetylpyridine (3-AP) treatment. A release of Asp and Glu, as well as a small but significant release of homocysteic acid (HCA) was confirmed in control rats. Climbing fiber deprivation by 3-AP treatment reduced the stimulated release of Asp by 48% in slices of cerebellar hemispheres, but not in vermis. Climbing fiber deprivation completely abolished the release of HCA in both hemispheres and vermis. The release of HCA, Asp, and Glu from slices of control and climbing fiber-deprived rats evoked by 50 mM K+ was greater than 90% Ca2(+)-dependent. These results support the hypothesis that Asp is a transmitter candidate of the climbing fibers projecting to the cerebellar hemispheres, but not to the vermis, and provide the first evidence that HCA can be linked to a specific pathway.