In humans, administration of recombinant human thyroid stimulating hormone (rhTSH) allows radioactive iodine dose reduction and higher efficacy in the treatment of multinodular goiter and thyroid cancer. A similar effect might be expected in dogs with thyroid carcinoma. However, if rhTSH leads to an increase in thyroid volume, it must be used carefully in dogs with large thyroid tumors to avoid compression of key anatomical structures. The aim of this study was to determine the short-term effect of rhTSH on thyroid volume and echogenicity, measured by ultrasonography, in healthy Beagles. Seven beagles were divided randomly in two groups in a prospective placebo-controlled blinded cross-over study. On day 1, one group received 100 μg of rhTSH intravenously and the other group received placebo. After a wash-out period of 3 weeks, the groups were crossed over. Evaluation of thyroid echogenicity, homogeneity, shape, capsule delineation, and measurement of thyroid length, width, and height were performed by the same observer at baseline, and at 6, 24, and 48 h after injection of rhTSH and placebo. There was no significant difference between the effect of rhTSH and placebo on thyroid volume (P=0.397), echogenicity, homogeneity, or capsule delineation. Time had a significant effect on thyroid volume (P=0.027). No adverse effects after rhTSH administration were noticed. The effect of rhTSH on thyroid volume in dogs with thyroid carcinoma needs to be assessed.