Artificial insemination (AI) involves the accurate detection of oestrus, the ability to pharmacologically induce ovulation, or both. This is particularly important in flying-foxes (Pteropus spp.) that do not have an overt behavioural oestrus. Pregnant mare serum gonadotropin (PMSG) stimulates folliculogenesis and ovulation in the Little Red Flying-fox (P. scapulatus; Towers PA and Martin L 1985 Proc. Aust. Soc. Reprod. Biol. 17, 115). In this study, a dose rate of 15 IU PMSG was used. Our study investigated if 15 IU PMSG would sufficiently induce ovulation in the larger, Black Flying-fox (P. alecto) or if a higher dose (30 IU) would be necessary. Before the mating season, a single injection of PMSG (Folligon^®, Intervet Australia Pty Ltd, Bendigo East, Australia) was administered i.m. to eight adult females at 15 IU (n = 4) or 30 IU (n = 4) on Day 0. On Day 4, semen was collected by electro-ejaculation from adult males and inseminated intravaginally into females under isoflurane (Forthane^®, Abbott Australasia Pty Ltd, Sydney, Australia) anaesthesia. Ovaries and reproductive tracts were surgically removed on Day 6, fixed, serially sectioned and stained using Gomori’s Stain. Histological sections were examined for evidence of ovarian activity and the presence of ova and spermatozoa in the reproductive tracts. Preliminary observations showed evidence of ovulation in both groups in the form of at least one CL in either ovary and an ovum in the ipsilateral oviduct or uterine horn. Ovaries of females stimulated with 30 IU PMSG differed noticeably from those treated with 15 IU in containing multiple, large, collapsed luteinized follicles with retained oocytes. Vascularization and glandular hypertrophy of the endometrium was also more evident in the higher dose group. No spermatozoa were observed in any of the excised tracts. These results suggest that whereas both doses of PMSG induce ovulation, the administration of 30 IU PMSG may over-stimulate the ovaries. This, in turn, could lead to an unphysiological environment for successful fertilization and embryonic development. A dose level of 15 IU or slightly above may be sufficient for subsequent attempts to stimulate folliculogenesis and ovulation in P. alecto. The absence of spermatozoa suggests that the site of insemination, the number of spermatozoa inseminated, or both requires further investigation if AI is to be successfully implemented in these species. We conclude that the ovarian responses to PMSG indicate that pharmacological induction of ovulation can be successfully achieved and thereby utilized in AI programs of endangered Pteropus species.