Mature bullseye puffer (Sphoeroides annulatus) fish were caught and taken into captivity. Fish with a mean oocyte diameter greater than 0.45 mm were divided equally into three experimental groups (n=11) that were treated with a saline injection (control), multiple injections of luteinizing hormone-releasing hormone analogue (LHRHa) (day 0—20 μg kg−1, day 2—40 μg kg−1, day 4—80 μg kg−1, day 6—160 μg kg−1) and a single administration of slow-release ethylene vinyl acetate (EVAc) co-polymer implants containing the identical LHRHa (day 0—75 μg per fish <800 g or 150 μg per fish >800 g). A total of 82% of the fish spawned in each of the two LHRHa groups (injection and implant) compared to just 18% in the control group. The majority of the fish treated with LHRHa, eight in the injection group and nine in the implant group, spawned during the first week after administration. There were no significant differences in mean egg size (group means 0.64–0.71 mm) and percentage fertilization (group means 90.2–97.3%) among the LHRHa-treated groups and when compared with the control fish that received saline injection and fish that ovulated naturally without any treatment. The successful spawning using LHRHa-based spawning protocols is an important step in the development of the culture of the bullseye puffer.