BACKGROUND: The neuropeptide kisspeptin and its receptor GPR54 play a crucial role in the reproductive function of mammals. Finding an efficient kisspeptin antagonist could be the first step towards a new method for non-surgical contraception in the dog. The aims of this study were to test kisspeptin antagonists 271 and 234 in vitro using flow-cytometry and to test p271 in vivo during different stages of the estrous cycle of the bitch. METHODS: In vitro: Chem-1 cells expressing the human GPR54 receptor were labeled with Fluo-3 AM, a fluorescence indicator for intracellular calcium, and examined by flow-cytometry. A concentration response study of hKP-10 and cKP-10 was designed to test if flow-cytometry is a reliable method for measuring intracellular calcium (as a measure for receptor activation) and to determine the optimal hKP-10 concentration. This data were then used to test the antagonists p234 and p271 in vitro. In vivo: P271 was tested during different stages of the estrous cycle of the bitch. In this study only the results in the follicular phase are discussed. Female dogs received 50 µg/kg/hour p271 by iv infusion for three hour. Two hours after the start of the infusion 0.5 µg/kg cKP-10 was iv administered. Controls received one iv injection of 0.5 µg/kg cKP-10. Blood samples before, during and after the peptide administration were collected for the determination of plasma [LH]. RESULTS: Both human and canine KP-10 gave a typical sigmoidal concentration-response curve in vitro. We found no antagonistic or agonist effect of peptide 234 and 271 in vitro. Furthermore, p271 was not able to reduce basal plasma LH concentrations and the LH response to cKP-10 in vivo in the bitch during follicular phase. CONCLUSION: P271 is probably not a good kisspeptin antagonist for the dog. Flow-cytometric calcium flux assays seem like a reliable method to study kisspeptin receptor activation in vitro. Advisors/Committee Members: Albers-Wolthers, C.H.J..