The colocalization of immunoreactivities similar to delta sleep inducing peptide (DSIP) and luteinizing hormone releasing hormone (LH-RH) was investigated by light and electron microscopic immunocytochemistry in the rat median eminence. At the light microscopic level, DSIP and LH-RH immunostained fibers, and varicosities exhibited a similar distribution pattern throughout the median eminence. Immunoreactive axons were mainly found in the lateral part of the external layer. Using an elution-restaining technique, the coexistence of LH-RH and DSIP immunoreactivities was observed in most labelled axons. To determine the intracellular localization of DSIP and LH-RH, we used double immunocytochemical labelling with species-specific antibodies and secondary antibodies conjugated to different sizes of gold particles. The two peptides were found colocalized in single axons. Immunoreactive terminals frequently showed direct membrane apposition with tanycyte processes but rare contacts with portal capillaries. No staining was observed in tanycytes and ependymal or glial elements. Moreover, we could demonstrate that LH-RH and DSIP (or a closely related molecular form) are contained not only in the same axons, but also in the same approximately 100-nm dense-core vesicles, suggesting cosecretion of these peptides.