With the intent to evaluate the efficiency of a contraceptive treatment for cyclic ovarian suppression in African Lionesses Panthera leo using a Gonadotrophin-Releasing Hormone (GnRH) agonist bioimplant, noninvasive fecal steroid assay associated with the observation of the behavioral estrus were employed for a period of 36 months. Five captive adult females, maintained with a vasectomized male, subcutaneously received a 9.4mg deslorelin acetate implant. The treatment initially stimulated behavioral estrus along with ovarian activity, demonstrated by an estrogen increase in two lionesses. A rise in progesterone concentration in two other animals suggested possible treatment-induced ovulation. After the initial period, deslorelin prevented ovarian activity for at least 22 months. Two females exhibited signs of behavioral estrus after 22 and 31 months. A third lioness with an increased estrogen concentration did not exhibit behavioral estrus signs or a consequent progesterone surge until 33 months after implantation, suggesting a possible resumption of ovarian activity. One female did not exhibit any behavioral estrus signs nor a rise in steroid levels after the “treatment-induced” estrus throughout the entire experiment (36 months). One lioness died after 15 months without exhibiting signs of estrus or an increased progesterone level, however, the estrogen concentration increased 12 months post-implantation, suggesting resumed ovarian activity. The study showed that long-term treatment with a GnRH agonist can be extremely effective as a contraceptive treatment in African lionesses, however, the duration of contraception may vary among individuals and may bear the risk of permanent loss of normal ovarian activity.