Alpha (α)- and beta (β)-adrenergic receptors in the bovine mammary gland are mainly present in the teat muscles and in the region where large milk ducts reach the cisternal cavities. The aim of the study was to test the hypothesis that the region of the large mammary ducts is the most important location of α- and β-adrenergic receptor stimulation affecting milk ejection and milk removal. Effects of α- and β-adrenergic receptor stimulation and of oxytocin (OT) receptor blockade on milking characteristics were tested in six cows. Milk flow was measured before and after the distal part of one teat, including the teat canal and teat sphincter, had been partly amputated. Before the operation, milk yield and peak flow rate decreased during α-adrenergic receptor stimulation and during the OT receptor blockade, and increased during β-adrenergic stimulation. After removal of the teat tip, relations of milk yield and peak flow rates after administration of α- and β-agonists and after application of an OT receptor blocking agent were similar to those before operation. Only total milk yield had decreased in the teat-amputated quarter owing to unhindered flow of cisternal milk before cluster attachment. Since responses to α- and β-adrenergic receptor stimulation as well as to OT receptor blockade do not differ with or without the teat sphincter, it is concluded that milk flow is mainly influenced by the muscle tone of the large mammary ducts.