The control of feral cats (Felis catus) in Australia is a key biological conservation issue. Male cats are more difficult to control than female cats. Collared and tagged female cats displaying estrous behavior have been considered as a way to lure male cats and reveal their locations. As female cats are seasonal breeders, artificial induction of estrous behavior following the administration of a long-acting estrogen could improve their use for this purpose. Estradiol cypionate was intramuscularly administered to nine entire non-pregnant female feral cats, of unknown estrous status, at 0.1, 0.3, or 0.5 mg/kg. Mean peak serum concentrations of estradiol-17β were 365 pg/mL (0.1 mg/kg), 1281 pg/mL (0.3 mg/kg), and 1447 pg/mL (0.5 mg/kg). The time-course of estradiol-17β concentrations after various doses of estradiol cypionate was assessed using non-compartmental and non-linear mixed-effects methods. At the highest-studied dose (0.5 mg/kg), the 50th percentile of estradiol-17β concentrations exceeded 0.1 ng/mL for 11.8 days, and 0.05 ng/mL for 14.6 days. The duration increased with increasing dose. No signs of toxicity were noticed in any cat during the study. This information will be useful to ongoing studies that are investigating ways to reduce the abundance of feral cats in Australia, especially adult male cats.