Multiple-unit recordings were obtained from the interstitial nucleus of Cajal, nucleus of Darkschewitsch and the superior colliculus of the cat during acquisition of classically conditioned pupillary dilation. Multiple-unit responses in all regions were enhanced by conditioning procedures. However, only the acquisition functions for the accessory oculomotor nuclei, i.e., interstitial nucleus of Cajal and nucleus of Darkschewitsch, were significantly correlated with the acquisition of conditioned pupillary dilation. These results were discussed in relation to the mechanism of autonomic control of conditioned pupillary dilation. It was concluded that inhibition of parasympathetic pupillomotor efferents via the accessory oculomotor nuclei may play a role in the acquisition of conditioned pupillary dilation.