Mating has profound effects on honey bee queen behavior and physiology. After mating, vitellogenesis is initiated and the egg-maturation is completed in the ovaries. Mated queens no longer leave the colony for mating flights, and initiate egg-laying behavior. Furthermore, there are dramatic differences in the pheromone profile of mated queens versus virgin queens, which leads to altered worker behavior. While workers virtually ignore virgin queens, they are attracted to mated queens and will form a retinue, where they surround the queen and lick and antennate her. It is not clear at which stage after mating the pheromone profile changes. Instrumental insemination produces many of the effects of mating, but the changes are substantially delayed. Furthermore, anecdotal evidence implies that single-drone inseminated queens are superseded much more rapidly in colonies, suggesting that workers react differently to inseminated queens, perhaps due to differences in pheromone profile. We monitored the latency to initiate egg-laying and the worker retinue response to queens after mating or insemination. We also compared the pheromone profiles of mated or inseminated queens two days after egg-laying began.