Trace eyeblink conditioning was investigated in 31 patients with focal cerebellar lesions and 19 age-matched controls. Twelve patients presented with lesions including the territory of the superior cerebellar artery (SCA). In 19 patients lesions were restricted to the territory of the posterior inferior cerebellar artery (PICA). A 3D magnetic resonance imaging was used to determine the extent of the cortical lesion and possible involvement of cerebellar nuclei. Eyeblink conditioning was performed using a 40 ms tone as conditioned stimulus (CS) followed by a stimulus free trace-interval of 400 ms and a 100 ms air-puff as unconditioned stimulus (US). In SCA patients with lesions including parts of the cerebellar interposed nucleus trace eyeblink conditioning was significantly impaired. Pure cortical lesions of the superior cerebellum were not sufficient to reduce acquisition of trace conditioned eyeblink responses. PICA patients were not impaired in trace eyeblink conditioning. Consistent with animal studies the findings of the present human lesion study suggest that, in addition to forebrain areas, the interposed nucleus is of importance in trace eyeblink conditioning. Although cortical cerebellar areas appear less important in trace compared with delay eyeblink conditioning, the present data strengthen the view that cerebellar structures contribute to different forms of eyeblink conditioning paradigms.