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Immunohistochemical distribution of [D-Ala2]deltorphin I (DADTI), a highly selective ligand for ƒÂ opioid receptors, was investigated in rat retina by using a specific antiserum. The antiserum against DADTI was raised in a rabbit by immunizing with a synthetic peptide (Tyr-D-Ala-Phe-Asp-Val-Val-Gly•ENH2) conjugated to poly-L-glutamate with water soluble carbodiimide. When examined by immunospot assay, the major epitope of the antiserum appeared to be in the C-terminal region containing Asp-Val-Val-Glu•ENH2, the sequence known to be responsible for opioid receptor selectivity. Immunohistochemically, DADTIlike-positive staining was found in some retinal amacrine cells situated in the inner nuclear layer. Their stained processes were distributed in the inner plexiform layer. In addition, a few positive cells, probably ectopic amacrine cells, were seen in the ganglion cell layer. Double immunostaining for DADTI and other neurotransmitters or neuropeptides revealed that 12.8% and 27.7% of DADTI-positive cells contained GABA and neuropeptide Y, respectively. However, DADTI-positive cells were seldom colocalized with somatostatin or tyrosine hydroxylase. The present study suggests that DADTI in retinal amacrine cells play role(s) functioning as an endogenous opioid peptide.