In the course of season 2010 (May–September), three 2-day trappings of female mosquitoes were carried out at two sites in order to determine the daily activity of the common mosquito species (e.g. species from genus Culex, Aedes, Ochlerotatus, Anopheles, etc.) in the area. CDC light traps filled with CO2 and placed at a height of 1 m were used to trap individuals, and were sampled every 2 h. A total of 19,604 female mosquitoes from 20 identifiable species were trapped: 7,549 at Sedlec and 12,055 at Kančí obora. The activity of the major species of mosquitoes in South Moravia differed throughout the course of the day. Calamity species of the genus Aedes and Ochlerotatus remained active throughout the day and night, but with different course. Aedes vexans, Ae. cinereus, and Ae. rossicus were most active in the late afternoon and highly active for most of the night. Ochlerotatus sticticus was captured most often in the afternoon, and its abundance decreased rapidly before sunset. The activity of Oc. cantans s.l. (Oc. cantans + Oc. annulipes) females varied little during the day and night. The daily activity for the main vectors of West Nile virus, Culex pipiens and Cx. modestus, were totally different from that of other species. Cx. pipiens females showed significant night activity, while Cx. modestus was most active in the evening. Nighttime activity was also observed in female mosquitoes of the genus Anopheles.