Complications of cervical myelography arising from the puncture of the subarachnoid space to collect the cerebrospinal fluid and to inject the contrast medium have been described in humans and animals. In this study, 2 ultrasound-guided procedures were developed for puncture of the atlanto-occipital subarachnoid space, collection of cerebrospinal fluid, and injection of contrast medium. Myelography was performed on 6 ataxic horses using these procedures. The first attempt to puncture the subarachnoid space was successful in 5 horses and in one horse, a second attempt was necessary. Collection of cerebrospinal fluid and injection of contrast medium were achieved without difficulty. Ultrasound-guided myelography allowed reduction of potential complications associated with blind percutaneous puncture of the subarachnoid space. Methods described in this study should be tried—at least initially in an experimental setting—to collect cerebrospinal fluid from the atlanto-occipital site in standing horses where it may represent an alternative method when lumbosacral cerebrospinal fluid collection has been unsuccessful or contaminated with blood.