The combination of a short-acting GnRH antagonist with a long-acting agonist could prevent the gonadotropin "flare-up" at the beginning of agonist treatment in other species. The objective of this study was to evaluate the efficacy and clinical safety of a single injection of the GnRH antagonist, acyline, to prevent post-GnRH agonist, deslorelin-estrous response in anestrous bitches. Eight, postpubertal, 18 to 35 kg, anestrous bitches were allocated to one of the following treatment groups: deslorelin acetate (Suprelorin®, Peptech), 10 mg sc (DA; n = 4) or deslorelin acetate (Suprelorin®, Peptech), 10 mg sc followed by acyline (NICHHD, NIH, USA) 330mg/kg sc 72 hs later (DA&ACY, n = 4). All the bitches were examined daily for detection of post-GnRH agonist estrous response and the appearance of systemic or local side effects related to the treatments for one month. In animals that presented an estrous response, progesterone (P4) serum determinations were carried out 3 wks after proestrus onset to test ovulation (P4 > 5 ng/ml). The frequency of bitches achieving estrous response, ovulation or side effects in each treatment group were analyzed by PROC FREQ. Days to estrous response were expressed as least squares means (LSM) ± SEM (SAS®). Estrous response did not vary between treatment groups (P > 0.05), as all (8/8) the animals presented it. Ovulation occurred in all DA bitches and in 3 of the 4 DA&ACY animals (P > 0.05). None of the bitches presented local or systemic side effect related to the treatments. Estrous response appeared 12.5 ± 0.1 and 5.2 ± 1.4 days after implantation in the DA&ACY and DA groups, respectively. It is concluded that, in this preliminary study, a single administration of the GnRH antagonist, acyline, failed to prevent post GnRH agonist stimulation in anestrous bitches. Further studies with repeated or higher doses of antagonists are necessary before antagonist depot formulations were available.