A detailed study of the activity of LHRH analog antagonists has been made in four assay systems which measure inhibition of the action of LHRH on isolated rat pituitaries in vitro, inhibition of the release of the LH induced by LHRH in vivo in adult male rats and adult male chimpanzees, and inhibition of spontaneous ovulation in cycling female rats. Only a partial correlation was observed between the in vitro and in vivo assays. Currently, the most potent LHRH analog antagonists in the present study were based on a 1,2,3,6-tetra-substituted LHRH sequence. The analogs [D less than Glu1,DPhe2,DTrp3,DTrp6]-LHRH, Ac-[Pro1,DPhe2,DTrp3,DTrp6]LHRH and [(Glu-Pro)1, dphe2,DTrp3,DTrp6]LHRH completely inhibited spontaneous ovulation in cycling rats at a dosage of 200 microgram/rat, sc. The most potent inhibitors of ovulation were always very potent in vitro, but other analogs having identical in vitro activities had little or no antiovulatory activity even at substantially higher dosages. The analogs inhibited the action of LHRH in the rat more easily than in the chimpanzee. Twelve of 13 analogs at the analog to LHRH ratio of 100:1 significantly inhibited the LH response, while only 5 of 9 of these same analogs inhibited the LH response in the chimpanzee at the analog to LHRH ratio of 333:1. Only 1 of 8 analogs at a high dosage inhibited the binding of labeled LH to the gonadal LH receptor in vitro. The inability of the less polar (cyclopentane carboxylic acid) analogs to inhibit ovulation could be explained, at least partially, in terms of impaired absorption sc. Although the cyclopentane carboxylic acid analogs effectively inhibited the action of LHRH in vitro and, when given iv in vivo, they were not effective in blocking the LHRH-stimulated LH response in adult male rats when given sc, which is the mode of administration of the antiovulatory assay, suggesting the importance of the route of administration.