If a queen runs out of sperm in her lifetime, new generations of queens will mate and produce their own colonies. The queen uses only a few of these sperm at a time in order to fertilize eggs throughout her life. However, only five to six million are stored within the queen's spermatheca. After several matings during this flight, a queen stores up to 100 million sperm within her oviducts. Virgin queens mate early in their lives and only attend one mating flight. Even drones that survive the mating flight are ejected from their nests, as they have served their sole purpose by mating. Male honey bees are only able to mate seven to 10 times during a mating flight, and after mating, a drone dies quickly, as his abdomen rips open when his endophallus is removed. The next male honey bee to mate with the queen will remove the previous endophallus and eventually lose his own after ejaculation. After ejaculation, a male honey bee pulls away from the queen, though his endophallus is ripped from his body, remaining attached to the newly fertilized queen.

A male drone will mount the queen and insert his endophallus, ejaculating semen. When a virgin queen flies to a site where thousands of male honey bees may be waiting, she mates with several males in flight.