For endangered species managed ex situ, production of offspring is a key factor to ensure healthy and self-sustaining populations. However, current breeding goals for the whooping crane (Grus americana) are impeded by poor reproduction. Our study sought to better understand mechanisms regulating ovarian function in ex situ managed whooping cranes and the regulatory function of the hypothalamic-pituitary-gonadal (HPG) axis in relation to follicle formation and egg laying. To characterize hormonal regulation of follicular development and ovulation, we collected weekly blood samples from six female whooping cranes during two breeding seasons, for a total of 11 reproductive cycles. The plasma samples were assessed for follicle stimulating hormone, luteinizing hormone, estradiol, and progesterone and the yolk precursors vitellogenin and very low-density lipoprotein. Ultrasonographic examination of the ovary was conducted at the time of blood collection. Preovulatory follicles (>12 mm) were present in laying cycles (n = 6) but absent in non-laying cycles (n = 5). The patterns of plasma hormone and yolk precursor concentrations corresponded to the stage of follicle development. Specifically, gonadotropin and yolk precursors concentrations increased as follicles transitioned from the non-yolky to yolky stage but did not increase further as the follicle advanced to preovulatory and ovulatory stages. Estrogen and progesterone concentrations increased as follicle size increased and reached peak concentrations (P < 0.05) when follicles developed to ovulatory and preovulatory stages, respectively. While overall mean circulating gonadotropin, progesterone, and yolk precursor concentrations did not differ for laying versus non-laying cycles, mean plasma estradiol in laying cycles was significantly higher than that in non-laying cycles. In summary, the findings suggested that disruption of mechanisms regulating follicle recruitment is likely responsible for the oviposition failure of the captive female whooping crane.