This study was carried out using Ixodes ricinus ticks collected during 2005 and 2006 from the Friuli Venezia Giulia (FVG) region in the northeastern part of Italy and an area along the Slovenian side of the western border of Italy. The results indicate that Rickettsia spp. is widely distributed throughout these areas, with the greatest prevalence in the central part of the FVG region. The prevalence of Rickettsia spp. was 4.5% during 2005 and 6.1% during 2006. By sequencing the 16S rRNA gene, we show for the first time the presence of Rickettsia helvetica in I. ricinus ticks in the FVG region and the presence of R. monacensis in ticks in both areas. Furthermore, we detected a sequence with a high homology with that of R. limoniae in a tick obtained from the alpine zone.