Background Ghrelin plays an important role in the regulation of food intake and body weight. It also decreases testosterone and opioid secretion. Objectives The goal of the present study was to investigate the effect of testosterone, morphine or simultaneous injection of testosterone and morphine on mean serum ghrelin concentration in sheep. Materials and Methods Ten sheep were divided into two groups (n = 5 in each group), they were fed with either 50 % or 100 % of their dietary energy needs for 10 days. Body weight was measured on the 1st and 10th day of the experiment. Animals in both groups received testosterone (60 μg/kg), morphine (0.15 mg/kg), or a simultaneous infusion of testosterone (60 μg/kg) and morphine (0.15 mg/kg), on the 8th, 9th, or 10th day of the experiment respectively. Blood samples were collected before and 2 hours after the infusions. Ghrelin concentration was determined by RIA (radio immunoassay). Results In the 50 % group, ghrelin concentrations increased significantly on the 8th day of the experiment, compared to the 1st day (P < 0.05). While in the 100 % group, no significant change was observed. In both groups the animals’ body weight did not increase significantly on the 10th day compared to the 1st day. Testosterone significantly increased ghrelin levels after injection compared to before infusion, in both groups (P < 0.05). Morphine increased ghrelin concentration in both groups, but this increase was not statistically significant. Simultaneous injection of testosterone and morphine together, significantly increased ghrelin concentration following injection compared to before infusion, in both groups (P < 0.05). Conclusions There is a direct correlation between food restriction, testosterone and ghrelin concentration in ruminants. However, a simultaneous injection of testosterone and morphine did not exert an additive effect on ghrelin secretion.