We have examined the effect of two GnRH antagonists, AcD-Nal1-Cl-D-Phe2-3-Pyr-D-Ala3- Arg5-D-Glu(AA)6-GnRH (NalGlu) and Ac3.4-dehydro-Pro1,-p-fluoro-D-Phe2, D-Trp3.6-GnRH (4pF), on in vivo and in vitro fertilization in rodents. Female rats were treated in the afternoon of proestrus with 2 μl of Nal-Glu or 4pF (0.5 and 5 mM) injected directly into one oviductal horn (experimental); saline was injected into the contralateral horn (control). Females were then mated and the oviducts were perfused for egg and sperm recovery. The results indicate that both antagonists inhibited in vivo fertilization. Thus, the percentage of fertilized eggs in control oviducts ranged from 92% ± 5% to 100% ± 0%, whereas in treated oviducts, fertilization ranged from 25% ± 6% to 73% ± 5%. GnRH antagonists did not interfere with the process of ovulation, sperm migration to the site of fertilization, or early embryo development. In additional experiments with mice, GnRH antagonists inhibited in vitro fertilization. One fertilization event that was specifically inhibited by GnRH antagonists was the process of sperm binding to the zona pellucida. This step was precisely monitored using the hemizona assay. GnRH antagonists did not affect sperm movement or acrosomal status. These observations indicated that local treatment with GnRH antagonists inhibit in vivo fertilization and give additional support to the idea that endogenous GnRH may play an important role during fertilization by increasing the efficiency of sperm-zona binding.