The insular cortex has been implicated in the reinforcing properties of opiates as well as in the integration of responses to sensory-motor stimulation. Moreover, the delta-opioid receptor (DOR) and the endogenous opioid ligand, Met5-enkephalin (ENK) are known to be prominently distributed in insular limbic cortex. To examine the anatomical sites for opioid activation of DOR in rat insular cortex, we used immunoperoxidase for detection of an antiserum raised against a peptide sequence unique to the DOR alone, and in combination with immunogold-silver labeling for ENK. Light microscopy showed intense DOR-like immunoreactivity (DOR-LI) in pyramidal cells and interneurons in deep laminae, and in varicose processes in both superficial and deep layers of the insular cortex. Ultrastructural analysis of layers V and VI in insular cortex showed that the most prominent immunoperoxidase labeling for DOR was in dendrites. This labeling was associated with asymmetric excitatory-type junctions postsynaptic to unlabeled terminals. Dendritic DOR-LI was also distributed along selective portions of non-synaptic plasma membranes and subsurface organelles. In dually labeled sections, dendrites containing DOR-LI sometimes received synaptic input from ENK-labeled terminals or more infrequently colocalized with ENK. Other axon terminals were exclusively immunolabeled for DOR or more rarely contained both DOR and ENK immunoreactivity. Within labeled axon terminals, distinct segments of the plasma membrane and membranes of immediately adjacent synaptic vesicles showed the largest accumulation of the peroxidase reaction product for DOR. These results indicate that in rat insular cortex DOR is primarily heteroreceptive, but also serves an autoreceptive function on certain ENK-containing neurons. Our results also provide the first ultrastructural evidence that in rat insular cortex endogenous opioids interact through the DOR (1) to modulate the postsynaptic responses to other excitatory afferents and (2) to presynaptically regulate the release of other neurotransmitters. The modulatory actions on both ENK-containing and non-ENK-containing neurons may contribute significantly to the reinforcing properties of exogenous opiates acting on the DOR in limbic cortex.