Mating nuclei populated with virgin queens and worker bees were sited at three mating stations in order to test and compare their isolation from drones. After 22 days, the nuclei were examined for the presence of brood, the spermatozoa stored in the queens’ spermathecae were counted, and the bodies and ovaries of the queens were weighed. The queens and 10 worker bees from each nucleus were also examined for Nosema spores. No queen produced brood at the Rog-Ponikve station, whilst 50% and 81% queens produced brood at the Čremošnjice and Semič stations, respectively. Spermathecae of queens from Rog-Ponikve station did not contain sperm. The average body weight of the queens from Rog-Ponikve, Čremošnjice and Semič, were 169.25, 200.25 and 203.61 mg, respectively and their average ovary weights were 21.00, 51.57 and 57.31 mg, respectively. Thus egg laying queens from the Čremošnjice and Semič mating stations were 1.2 x heavier and their ovaries were 2.7 and 2.4 x heavier respectively, than those of the non ovipositing queens from the Rog-Ponikve station (P<0.001). No significant differences were found in the average number of Nosema spores (P>0.05) in the worker bees from the 3 stations and no spores were detected in any of the queens. The experiment showed that the Rog-Ponikve mating station has a secure level of drone isolation. The importance of testing the isolation of mating stations is discussed in this study.