The relationships between delta sleep-inducing peptide (DSIP) and GnRH immunoreactivity within the guinea pig median eminence are investigated by light and electron microscopic immunocytochemistry. Indirect immunofluorescence and elution-restaining experiments show that at the light microscopic level the distribution patterns of DSIP and GnRH immunoreactivity are indistinguishable. This suggested the possible coexistence of both immunoreactivities within the same fibers and neurosecretory endings. At the electron microscopic level, a preembedding double-immunolabeling technique using both indirect immunoperoxidase and immunogold methods, clearly indicate that DSIP and GnRH immunoreactivity are frequently colocalized within single secretory granules. In addition DSIP/GnRH immunoreactive nerve endings were also observed often in close proximity to tanycyte elements. Taken together, the present results provide for the first time ultrastructural evidence for the presence of DSIP immunoreactivity and demonstrate that DSIP and GnRH immunoreactivities may be coexpressed within the same neuronal elements in the median eminence.