Many rickettsiae of the spotted fever group are emerging pathogens causing serious diseases associated with vertebrate hosts. Ixodidae ticks are known as their vectors. Investigation of the relative abundance of questing Ixodes ricinus and their infection with Rickettsia spp. in an agricultural site comprising a game reserve in Slovakia was the aim of this study. In total, 2198 I. ricinus (492 larvae, 1503 nymphs and 203 adults) were collected by flagging the vegetation along 100 m(2) transects in Rozhanovce (eastern Slovakia): 334, 595 and 1269 in 2011, 2012 and 2013, respectively. Considering questing nymphs and adults, the highest relative density of 81 individuals/100 m(2) was observed in May 2013, the lowest of 0.3 individuals/100 m(2) in March 2012. A total of 1056 ticks (853 nymphs, 100 females and 103 males; 2011: n = 329, 2012: n = 509 and 2013: n = 218) were individually screened by PCR-based methods for the presence of Rickettsia spp. The overall prevalences were 7.3 % for nymphs, 15 % for females, 7.8 % for males; 7.0 % in 2011, 8.4 % in 2012, and 8.7 % in 2013. The maximum prevalences were observed in July in nymphs and in May in adults. Sequencing showed infection with R. helvetica in 73 ticks (72.6 % nymphs, 16.4 % females, 11 % males) and with R. monacensis in 11 ticks (8 nymphs, 3 females). The results showed the circulation of pathogenic Rickettsia species in the agricultural site and a potential risk for humans to encounter infected ticks.