1. The actions of the endogenous excitatory amino acids (EAAS) glutamate (Glu), aspartate (Asp) and homocysteate (HCA) on Purkinje cells and neurones of the deep nuclei in cerebellar slice cultures were investigated using intracellular recordings in the single-electrode voltage-clamp mode and the whole-cell configuration of the patch-clamp technique. 2. Purkinje cells and neurones of deep cerebellar nuclei were identified according to their localization in the living cultures, their morphology as revealed by intracellular injections of Lucifer Yellow and their immunoreactivity to antibodies to the 28 kDa Ca2(+)-binding protein. 3. When Purkinje cells were voltage-clamped near their resting membrane potential in a TTX-containing salt solution, Glu, Asp and HCA induced inward currents which were abolished by 6-cyano-7-nitroxaline-2,3-dione (CNQX), a selective antagonist of the non-N-methyl-D-aspartate (NMDA) subtype of EAA receptors. The selective antagonist of NMDA receptors, D-(-)-2-amino-5-phosphonovaleric acid (D-APV), was ineffective in blocking the responses induced by these three amino acids. NMDA, even at high concentrations and in magnesium-free bathing solution, had no detectable effect on membrane properties of Purkinje cells grown in culture during 11-34 days. 4. In magnesium-containing saline, the amplitude of the responses induced by Glu, Asp and HCA was a linear function of the membrane potential. 5. In contrast, neurones of the deep cerebellar nuclei were responsive to NMDA and the inward currents induced by Glu, Asp and HCA were partially blocked both by CNQX and by D-APV. 6. In magnesium-containing saline, the amplitude of the currents induced by NMDA as well as by the three endogenous EAAs decreased at hyperpolarizing holding potentials whereas the current-voltage relation of the responses induced by quisqualate (QA) was strictly linear. 7. It is concluded that Purkinje cells in cerebellar slice cultures do not express NMDA receptors and that excitation of these neurones by the endogenous amino acids Glu, Asp and HCA is mediated exclusively through the activation of non-NMDA receptors. In the same preparation, neurones of the deep cerebellar nuclei possess NMDA and non-NMDA receptors which can be both activated by the three endogenous excitatory amino acids.