Bilateral simultaneous sampling of the inferior petrosal sinuses is an extremely sensitive, specific, and accurate test for diagnosing Cushing disease and distinguishing between that entity and the ectopic ACTH syndrome. It is also valuable for lateralizing small hormone-producing adenomas within the pituitary gland. The inferior petrosal sinuses connect the cavernous sinuses with the ipsilateral internal jugular veins. The anatomy of the anastomoses between the inferior petrosal sinus, the internal jugular vein, and the venous plexuses at the base of the skull varies, but it is almost always possible to catheterize the inferior petrosal sinus. In addition, variations in size and anatomy are often present between the two inferior petrosal sinuses in a patient. Advance preparation is required for petrosal sinus sampling. Teamwork is a critical element, and each member of the staff should know what he or she will be doing during the procedure. The samples must be properly labeled, processed, and stored. Specific needles, guide wires, and catheters are recommended for this procedure. The procedure is performed with specific attention to the three areas of potential technical difficulty: catheterization of the common femoral veins, crossing the valve at the base of the left internal jugular vein, and selective catheterization of the inferior petrosal sinuses. There are specific methods for dealing with each of these areas. The sine qua non of correct catheter position in the inferior petrosal sinus is demonstration of reflux of contrast material into the ipsilateral cavernous sinus. Images must always be obtained to document correct catheter position. Special attention must be paid to two points to prevent potential complications: The patient must be given an adequate dose of heparin, and injection of contrast material into the inferior petrosal sinuses and surrounding veins must be done gently and carefully. When the procedure is performed as outlined, both inferior petrosal sinuses can be catheterized in more than 98% of patients. The complication rate is low, and the theoretical risk of major morbidity or death is less than 1% (neither has yet occurred, to our knowledge). The most common complication is groin hematoma.