The mu-opioid receptor MOR1 is present on primary afferent axons and a population of neurons in the superficial dorsal horn of the rat spinal cord. In order to determine which types of neuron possess the receptor we carried out pre-embedding immunocytochemistry with antibody to MOR1 and combined this with a post-embedding method to detect GABA and glycine in the rat. MOR1 immunoreactivity was seen on many small neurons in lamina II and a few in the dorsal part of lamina III. Although immunostaining was mainly restricted to the cell bodies and dendrites of these neurons, in some cases it was possible to see their axons, and a few of these entered lamina III. One hundred and thirty-nine MOR1-immunoreactive cells were tested with GABA and glycine antibodies, and the great majority of these (131 of 139; 94%) were not GABA or glycine immunoreactive, while the remainder showed GABA but not glycine immunoreactivity. These results suggest that most of the cells in the superficial dorsal horn which possess MOR1 are excitatory interneurons. They support the hypothesis that part of the action of mu-opioid agonists, such as morphine, involves the inhibition of excitatory interneurons which convey input from nociceptors to neurons in the deep dorsal horn, thus interrupting the flow of nociceptive information through polysynaptic pathways in the spinal cord.