The participation of the rostral-dorsal part of the posterior interpositus nucleus in the genesis of classically-conditioned eyelid responses in alert cats was studied. Cats were prepared for the recording of left eyelid movements with the search-coil-in-a-magnetic-field technique and of the electromyographic activity of the orbicularis oculi muscle. A stimulating electrode was implanted in the contralateral red nucleus and a guide cannula in the ipsilateral interpositus. The cannula allowed unitary recordings, electrical microstimulation, drug injection, and push-pull perfusion of the selected interpositus site. Perfusate was used for amino acid analysis with an HPLC-EC method. Animals were conditioned with a delay (CS: tone, US: air puff) paradigm. Antidromically identified interpositus neurons fired during eyelid conditioned responses (CRs) with a variable delay to CR start. Some neurons (type A) increased and other (type B) decreased their firing during CR performance. Electrical microstimulation of posterior interpositus was able to increase CR amplitude when applied during CS presentation. Muscimol decreased CR amplitude in well-trained animals. The analysis of push-pull perfusate showed a significant increase in glutamate, glycine, and taurine, but not GABA levels, during conditioning in comparison with habituation and extinction periods. Results indicate an involvement of the rostral-dorsal part of the posterior interpositus nucleus in the genesis of eyelid conditioned responses, playing an enhancing or controlling role in CR performance, rather than being its initiator.