A novel member of the vasopressin/oxytocin superfamily, octopressin (OP), has been isolated from Octopus vulgaris. Since another peptide of this superfamily, cephalotocin (CT), was isolated from the same species [Neurosci. Lett. 134 (1992) 191], Octopus has two members of the superfamily as in vertebrates, an observation made for the first time in invertebrates. Octopressin caused contractions of the Octopus peripheral tissues such as oviduct, aorta, rectum, etc. Cephalotocin had no effects on tested tissues. The octopressin and cephalotocin precursors were composed of a signal peptide, a nonapeptide, and a neurophysin domain-the typical structural organizations of the superfamily precursors. Reverse transcription polymerase chain reaction (RT-PCR)/Southern blot analysis revealed that octopressin mRNA was expressed in the supraesophageal and subesophageal brains, and the buccal and gastric ganglia. Cephalotocin mRNA was expressed mostly in the subesophageal brain. In situ hybridization in the brain showed that octopressin mRNA was localized in many lobes. Expression of cephalotocin mRNA was almost limited in the ventral median vasomotor lobe. Some of the neurons in this lobe are the source of the neurosecretory system of the vena cava, where cephalotocin was originally isolated. These results suggest that octopressin may be a multifunctional neuropeptide contributing to reproduction, cardiac circulation, and feeding. Cephalotocin may play important roles in metabolism, homeostasis, etc., as a neurohormone.