With the use of the unlabeled antibody enzyme technique and antiserum to luteinizing hormone-releasing hormone (LHRH), the distribution of LHRH in the adult male guinea pig was studied. Immunoreactive deposits were found in the cell bodies of hypothalamic neurons in the medial preoptic, anterior hypothalamic, suprachiasmatic, and arcuate nuclei. Ten to thirty LHRH-containing perikarya were seen per brain. Four fiber tracts positive for LHRH were described: Tract I runs on the pre-commissural side of the anterior commissure from the bed nucleus of the stria terminalis and medial preoptic area to the suprachismatic nucleus: Tract II runs on the post-commissural side to the retrochiasmatic portion of the suprachiasmatic nucleus; Tract III originates in the arcuate nucleus and the ventral portion of the ventromedial nucleus and courses medially and caudally to enter the median eminence; Tract IV travels from the mammillary bodies to the mid-brain. Three weeks after surgical isolation of the medial basal hypothalamus there was a slight decrease in the amount of LHRH in the median eminence. These data suggest that LHRH is synthesized in more than one hypothalamic nuclear group but that the majority of axons containing the hormone in the median eminence originate in the medial basal hypothalamus.