A total of 691 Ixodes ricinus (22 male, 39 female, 501 nymphs and 129 larvae), the tick vector of Lyme borreliosis, were collected by flagging from vegetation in 11 areas at altitudes between 789 m and 1350 m above sea level in mixed woodland with pasture land (cattle) in the province of Styria in Austria. The ticks were individually examined for presence of Borrelia burgdorferi sensu lato by dark-field microscopy and 107 of them by real-time PCR. Attempts to cultivate borreliae were made in BSK-H medium. The overall positivity rate of all collected ticks (excepting larvae) was 10.9%: 9.1% in males, 17.9% in females and 10.4% in nymphs. The 129 larvae examined showed no presence of B. burgdorferi s.l. The mean infection rate of I. ricinus collected at the highest altitude in this study, Gaberl at 1350 m a.s.l. - and at the same time the highest one reported in Europe – was 6.4%: 1/9 males, 2/18 females and 6/114 (5.3%) nymphs were positive. Culture attempts were positive in 12 cases and species identification showed eight isolates were B. afzelii and four B. garinii. Three additional positive results found by PCR (negative by culture) were identified twice as B. afzelii and once as B. garinii. This study shows that the risk of acquiring Lyme borreliosis in habitats at higher altitudes is limited, because of the lower density of I. ricinus and its lesser infection rate than at lower altitudes in central Europe, but nevertheless the risk does exist.