The hypothalamic decapeptide, GnRH, stimulates LH and FSH release from pituitary gonadotrophs. Many synthetic peptide analogs, both agonist (GnRH-A) and antagonist (GnRH-AT), have been developed which bind specifically to the GnRH receptor. We have utilized highly potent GnRH-A and GnRH-AT analogs labeled with 18 nm colloidal gold to analyze ultrastructurally the events of binding and interiorization of these specific ligands by gonadotrophs in vitro. To examine internalization of GnRH-A-gold, gonadotrophs were cooled to 4 degrees C and equilibrated with the ligand for 1 h. Next, the cells were either fixed immediately or warmed to 37 degrees C for various times (5, 15 and 30 min) and prepared for electron microscopy. For GnRH-AT-gold, which binds slowly at 4 degrees C, the ligand was incubated with gonadotrophs at 37 degrees C for 15, 30 and 60 min, and the cells were processed for electron microscopy at each time point. In both cases, control gonadotrophs were also incubated in an excess of GnRH-A and GnRH-AT, respectively, in the presence of the gold-conjugated ligands. The results indicated that GnRH-A-gold was bound and rapidly internalized via a receptor-mediated endocytic pathway. GnRH-AT-gold was also bound but showed only limited entry into gonadotrophs; the percentage of intracellular GnRH-AT-gold in gonadotrophs was the same as in other pituitary cells contaminating the gonadotroph fraction and did not increase with time. In the gonadotroph, binding of the specific antagonist ligand to GnRH receptors does not stimulate its interiorization, in contrast to the rapid endocytosis and processing of the agonist ligand. These data suggest that specific ligand internalization requires prior receptor activation, and that GnRH-AT which does not activate the receptor remains bound at the cell surface for a prolonged period.