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Delta-sleep-inducing peptide (DSIP) colocalizes within gonadotropin-releasing-hormone (GnRH)-containing neurons in adult hypothalamus and could play a role in the regulation of hypothalamic-pituitary axis in adults. To support the possibility that DSIP also participates in fetal neuroendocrine events and to demonstrate the ontogenic evidence of coexisting neuropeptides, we have performed a detailed immunocytochemical study of DSIP- and GnRH-immunoreactivity in fetal hypothalamus of guinea pig. Using indirect immunofluorescent and sequential double-immunolabeling (elution-restaining) techniques, the results indicated that DSIP immunoreactivity was initially detected at the 38th day of gestation. In contrast to the first appearance of GnRH immunoreactivity at day 28, therefore, a 10-day delay was found. Such a delay remains as yet unexplained. From its first occurrence, DSIP immunoreactivity was always labeled with GnRH, whereas some of GnRH-immunoreactive structures did not display a DSIP immunoreactivity. But with the growth of fetus, especially before and after birth, a complete overlap between DSIP and GnRH immunoreactivity was observed throughout various regions of hypothalamus. Attention was also focused on prenatal morphological development of DSIP/GnRH- and GnRH-immunolabeled neurons. Initially, labeled neurons were visualized as uni- or bipolar types. Thereafter, their smooth and irregular subtypes could be distinguished. During later fetal age, relatively mature features were evident such as the increase of multipolar and irregularly labeled neurons. Taken together, these data provide, for the first time, anatomical evidence that DSIP exists in fetal hypothalamus and that, like GnRH, it could regulate the hypothalamic-pituitary axis during ontogenesis.