Regular replacement of old and problematic queen bees is necessary to maintain the strength and pro- ductivity of bee colonies. Beekeepers replace queen bees using queen cells, virgin queens, or mated laying queens. In this study, we explored factors influencing the acceptance of the virgin queens. A comprehensive field study was conducted in the summer of 2023, involving the introduction of 754 virgin queens of different age and 194 queen cells into mating nuclei of various states using three different methods. The article aims to provide recommen- dations to beekeepers for introducing virgin queens, thereby increasing the frequency of successful requeening. The duration of the colony being queenless has a significant impact on the acceptance of queens. If the colony is prepared in advance, at least five days prior, immature and mature virgin queens will be accepted equally well (60‒77%). Conversely, suppose queen replacement is done without preparation for a colony that has been queen- less for one day only. In that case, the result will be significantly lower due to the short queenless period (41%), and the age factor of queen bees will come into play. If queen replacement is done with preparation, the chosen timing within the season will play a significant role, as weather conditions can influence acceptance and mating, varying from 48% to 89%. Suppose a bee colony has long lost its queen and has developed laying worker bees. In that case, there is still a 55% chance of successfully introducing a virgin queen into such a colony without using time-consuming methods.