To understand the spawning ecology of the Japanese eel, the spawning time of this species was estimated based on measurements of the ascending speed of eggs and previously obtained data. Two types of water temperature parameters were calculated assuming an arbitrary spawning time. The 'incubation temperature' of 53 eggs collected in the spawning area was estimated based on the developmental stage of each egg and experimentally determined relationships between water temperature and incubation duration. The 'experienced temperature' of eggs ascending in the water column after spawning was estimated based on an ascending egg speed of 3.69 m/h and spawning depth of 230 m determined from a pop-up satellite archival tag release experiment on silver eels conducted in the same area. The incubation and experienced temperatures of the eggs coincided only at 20:20-22:30 h, 3 days prior to the new moon. This period is only a few hours after the diel vertical migration of Japanese eels in the evening, when adults move up from a depth of ~800 m (approximately 5 °C) to shallower waters of 200-250 m depth (approximately 20 °C). Our findings will facilitate improvements in aquaculture techniques and the detection of eel spawning events in the open ocean.