The 29/30 amino acid neuropeptide galanin has been implicated in pain processing at the spinal level and local dorsal horn neurons expressing the Gal(1) receptor may play a critical role. In order to determine the transmitter identity of these neurons, we used immunohistochemistry and antibodies against the Gal(1) receptor and the three vesicular glutamate transporters (VGLUTs), as well as in situ hybridization, to explore a possible glutamatergic phenotype. Gal(1) protein, which could not be demonstrated in Gal(1) knockout mice, colocalized with VGLUT2 protein, but not with glutamate decarboxylase, in many nerve endings in lamina II. Moreover, Gal(1) and VGLUT2 transcripts were often found in the same cell bodies in laminae I-IV. Gal(1)-protein and galanin-peptide showed an overlapping distribution but were not colocalized. Gal(1) staining did not appear to be affected by dorsal rhizotomy. Taken together, these findings provide strong evidence that Gal(1) is a heteroreceptor expressed on excitatory glutamatergic dorsal horn interneurons. Activation of such Gal(1) receptors may thus decrease the inhibitory tone in the superficial dorsal horn, and possibly cause antinociception.