A stereotaxic apparatus for access to the lateral brain ventricle of Holstein cows was constructed. This apparatus permits topographic orientation on the head of an anesthetized cow that has been placed in right lateral recumbency. After assessing the XY-plane in parallel to the frontal bone, correct coordinates in reference to the skullcap were adjusted. The lateral brain ventricles were accessed by drilling holes perpendicular to the XY-plane. The subsequent surgical implant of a tailor-made cannula guide enabled the repeatable placement of a catheter. This permanent catheter allowed the frequent withdrawal of cerebrospinal fluid from and injections into cerebral ventricles of nonanesthetized dairy cows. This method is critical for elucidating the role of central hormones, metabolites, and electrolytes in the regulation of diverse physiological processes and for the study of neuropathological disorders in cows.