The purpose of this study was to evaluate if freezing-thawing and cooling processes affect the structural properties and biological activity of commercial equine chorionic gonadotropin (eCG). First, the structure profile of diluted eCG underwent none, one or three cycles of freezing-thawing was analysed by reverse phase high-performance liquid chromatography (RP-HPLC). In a second experiment, groups of prepuberal rats were treated with sterile water for injection USP or eCG that underwent none, one or three cycles of freezing-thawing to assess the increase of ovarian weigh. Finally, groups of prepubertal gilts were treated with diluted eCG immediately after reconstitution (T1), after refrigeration for six months (T2) and after freezing and subsequently thawing for one (T3) or three (T4) cycles. The control group (T5) received sterile water for injection USP without eCG. Ovulation was induced with human chorionic gonadotropin (hCG), administered 72 h after the eCG. Gilts were slaughtered five days after the hCG injection and ovaries were recovered and analysed for the presence of corpora lutea. Data were analysed by ANOVA and Fisher’s exact tests. In the analyses by RP-HPLC, the retention times of cold stressed eCG were similar to unstressed control. The mean ovarian weight of rats treated with cold stressed and unstressed eCG was statistically higher than water control (P < 0.05). Lastly, significantly more gilts ovulated in groups T1, T2, T3 and T4 than in the control T5 (P < 0.05). It was concluded that freezingthawing, as well as cooling over a period of up to six months, did not significantly change the structural properties or biological activity of eCG.