Ixodes ricinus ticks were collected from dragging vegetation and from shot roe deer in the province of Trento and Belluno in northern Italy. Ticks were pooled for analyses and from 1060 pools of ticks collected in the province of Belluno and 12390 tick samples collected in Trentino, four proved positive by immunofluorescence microscopy using a tick-borne encephalitis (TBE)-specific antiserum. The identity of the virus isolates was determined by RT-PCR cycle sequencing and they were all found to be closely similar (> 98% nucleotide identity) to typical western European TBE complex viruses as found in Austria. The isolates from Trentino differed from the Neudorfl strain of western European TBE virus at eight nucleotide positions but as these nucleotide substitutions were all synonymous, there were no amino acid changes. These results imply that the virus isolates in Trentino have changed slightly from the typical European strains isolated in nearby Austria. The abundance of questing ticks and ticks feeding on roe deer was greater in TBE positive hunting districts than in hunting districts where TBE complex viruses were only probable or believed to be absent. In TBE positive and probable districts synchrony in the seasonal dynamics of larvae and nymphs of L. ricinus was observed. This study provides evidence to suggest that roe deer may have an important role to play in the maintenance of tick density and in the persistence of TBE virus.