The objective of this study was to describe testosterone (T) response to GnRH challenge in antagonist-treated dogs over a 30-day period. Eight mongrel dogs were randomly assigned to either the GnRH antagonist acyline 330 μg/kg sc (ACY; n = 4) or a placebo group (PLA; n = 4). The dogs were serially challenged with the GnRH agonist, buserelin 0.2 μg/kg sc on days -1, 1, 3, 7, 10, 14, 21 and 30. On these days, blood samples for T determinations were collected before (-30 min) and 60, 120 and 180 min after the agonist injection. Basal (-30 min) and post-GnRH agonist stimulation T values were compared by anova for repeated measures. Before treatments (day -1), there were no differences in basal T serum concentrations between groups (p > 0.1). After treatments, basal T showed a significant interaction between treatment and day (p < 0.05). Furthermore, when both groups were analysed independently, basal T varied in the ACY (p < 0.01) but not in the PLA group (p > 0.1). On day -1, before treatments, the stimulation tests had only a time effect (p = 0.05) although on days 1 (p < 0.01), 3 (p < 0.01), 7 (p < 0.01), 10 (p < 0.01) and 14 (p < 0.05), the response to the agonist differed between groups, becoming similar on days 21 (p > 0.05) and 30 (p > 0.05). It was concluded that, in dogs, a single administration of the GnRH antagonist prevented canine gonadal axis to physiologically respond to agonistic challenge during 14 days.