It has been reported that transections of the fimbria-fornix or lesions of the hippocampus selectively impair spatial working memory. Disruptive effects of these lesions on reference memory performance, however, have also been reported. We studied the effects of selective fimbria lesions on the acquisition of a complex spatial discrimination in the cone field. The cone field task is a place learning task that permits the simultaneous assessment of working and reference memory performance. Reproducible bilateral stereotaxic lesions were made by knife-cuts parallel to the midline. Sham lesions consisted of similar knife-cuts that were restricted to the overlying tissue. The rats were randomly started from 1 of 4 positions in order to prevent the development of a fixed food search pattern. On both memory components, fimbria-lesioned rats made about twice as many errors as the sham-lesioned and intact subjects, even after extensive training. Transection of the fimbria caused pronounced cholinergic denervation, predominantly at the more ventral part of the hippocampus, as indicated by reduced acetylcholinesterase histochemistry. Our results suggest a major role of the cholinergic innervation of the ventral hippocampus in spatial discrimination.