Uptake and retention of GnRH by pituitary was compared to that of GnRH-ethylamide (GnRH-EA) and D-Ala6-GnRH-ethylamide (D-Ala6-GnRH-EA) to determine if differences in these parameters might partially account for the increased biopotency of these superactive analogs. Ovariectomized estrogen/progesterone-treated rats were given an intracarotid injection of either 125I-labeled GnRH, -GnRH-EA, or -D-Ala6-GnRH-EA. Maximum uptake occurred at 2 min for GnRH (8000 cpm/pit), 5 min for GnRH-EA (10 000 cpm/pit), and 45 min for D-Ala6-GnRH-EA (24 000 cpm/pit). Thereafter pituitary content decreased out to 1, 2, and 4 h, resp. Specific uptake of all 3 peptides was shown autoradiographically to be restricted only to immunostained gonadotrophs with the silver grains being localized intracellularly at all time points studied. Serum concentrations of LH increased in response to all 3 peptides in proportion to their uptake and did not begin to decrease until after maximum uptake of each peptide had occurred. Thus, the extent of uptake and length of retention of these peptides in the pituitary correlate well with their relative biopotencies, D-Ala6-GnRH-EA greater than GnRH-EA greater than GnRH.