In a countrywide investigation of the ecological factors that contribute to Lyme borreliosis risk, a longitudinal study on population dynamics of the sheep tick Ixodes ricinus and their infections with Borrelia burgdorferi sensu lato (s.l.) was undertaken at 24 sites in The Netherlands from July 2006 to December 2007. Study sites were mature forests, dune vegetations, or new forests on land reclaimed from the sea. Ticks were sampled monthly and nymphal ticks were investigated for the presence of Borrelia spp. I. ricinus was the only tick species found. Ticks were found in all sites, but with significant spatial and temporal variations in density between sites. Peak densities were found in July and August, with lowest tick numbers collected in December and January. In some sites, questing activities of I. ricinus nymphs and adults were observed in the winter months. Mean monthly Borrelia infections in nymphs varied from 0% to 29.0% (range: 0%-60%), and several sites had significantly higher mean nymphal Borrelia infections than others. Four genospecies of Borrelia burgdorferi s.l. were found, with B. afzelii being dominant at most sites. Borrelia infection rates in nymphal ticks collected in July, September, and November 2006 were significantly higher (23.7%, p<0.01) than those in the corresponding months of 2007 (9.9%). The diversity in Borrelia genospecies between sites was significantly different (p<0.001). Habitat structure (tree cover) was an effective discriminant parameter in the determination of Borrelia infection risk, as measured by the proportion of nymphal ticks infected with B. burgdorferi s.l. Thickness of the litter layer and moss cover were positively related to nymphal and adult tick densities. The study shows that Borrelia-infected ticks are present in many forest and dune areas in The Netherlands and suggests that in such biotopes, which are used for a wide variety of recreational activities, the infection risk is high.