We have developed RIAs using antisera directed against the cryptic peptide of the GnRH precursor (termed GnRH-associated peptide, GAP) and have used these together with a GnRH assay to characterize proGnRH-derived peptides in rat hypothalamic extracts. On Sephadex chromatography we have identified three molecular forms of GAP-like immunoreactivity (GAP-LI), in addition to the GnRH decapeptide. The largest of these forms is an 8.0-kilodalton (kD) GAP-LI which appears to be the complete proGnRH peptide. The second is a 6.5-kD GAP-LI, and is similar to the complete cryptic peptide (i.e. proGnRH14–69 or GAP1–56). The third peptide is a 2.5 kD C-terminal fragment of the cryptic peptide, representing a processed form of GAP. In whole hypothalamic extracts from normal rats the 8.0-kD form was the major form, comprising 60–70% of the total GAP-LI. All three forms were present in three distinct areas of the rat hypothalamus, namely median eminence (ME), anterior and mid-hypothalamus. However in the ME the proportion of 8.0-kD GAP-LI was significantly reduced and the proportion of 6.5-kD GAP-LI significantly increased compared to anterior and mid-hypothalamic samples (p < 0.05). In whole hypothalamic extracts from pregnant and lactating rats the total content of proGnRH-derived peptides was reduced but the relative proportions of these peptides were not significantly changed from normal female rats. However in postlactating rats, 2 weeks after removal of pups, the total levels of GAP-LI were unchanged compared to normals, but the percentage of 8.0-kD GAP-LI was significantly decreased and the percentage of 2.5-kD GAP-LI significantly increased compared to normals (p < 0.05), suggesting that proGnRH may undergo additional processing dependent on physiological condition. In fetal and neonatal rats the proportion of the 6.5-kD peptide was increased and that of the 8.0-kD peptide decreased compared to adults, and this change became less significant with increasing age. In ovariectomized rats the proportion of 6.5-kD GAP-LI was increased and that of 8.0-kD GAP-LI decreased; this change was partially reversed with steroid treatment. Both the 6.5 and 2.5-kD forms were released by high K+ stimulation of neonatal hypothalamic cells in culture. These results indicate that there is differential processing of the proGnRH precursor within the hypothalamus and in altered physiological s