To characterize neuronal pathways that release opioid peptides in the rat dorsal horn, multiple-label immunohistochemistry, confocal microscopy, and computerized co-localization measures were used to characterize opioid-containing terminals and cells. An antibody that selectively recognized beta-endorphin labeled fibers and neurons in the ventral horn as well as fibers in the lateral funiculus and lamina X, but practically no fibers in the dorsal horn. An anti-enkephalin antibody, which recognized Leu-, Met-, and Phe-Arg-Met-enkephalin, labeled the dorsolateral funiculus and numerous puncta in laminae I-III and V of the dorsal horn. An antibody against Phe-Arg-Met-enkephalin, which did not recognize Leu- and Met-enkephalin, labeled the same puncta. Antibodies against dynorphin and prodynorphin labeled puncta and fibers in laminae I, II, and V, as well as some fibers in the rest of the dorsal horn. Dynorphin and prodynorphin immunoreactivities colocalized in some puncta and fibers, but the prodynorphin antibody additionally labeled cell bodies. There was no co-localization of dynorphin (or prodynorphin) with enkephalin (or Phe-Arg-Met-enkephalin). Enkephalin immunoreactivity did not colocalize with the C-fiber markers calcitonin gene-related peptide (CGRP), substance P, and isolectin B4. In contrast, there was some colocalization of dynorphin and prodynorphin with CGRP and substance P, but not with isolectin B4. Both enkephalin and dynorphin partly colocalized with vesicular glutamate transporter 2, a marker of glutamatergic terminals. The prodynorphin-positive neurons in the dorsal horn were distinct from neurons expressing mu-opioid receptors, neurokinin 1 receptors, and protein kinase C-gamma. These results show that enkephalins and dynorphins are present in different populations of dorsal horn neurons. In addition, dynorphin is present in some C-fibers.