Serum samples were collected from healthy blood donors in 5 regions in Sweden in 1999, i.e. from the local Blood Centres (collecting facilities) in Boden, Jönköping, Lund, Skövde, and Uppsala. In total, 498 serum samples (63% males, 37% females) were received and tested by immunofluorescence assay for antibodies against B. elizabethae, B. grahamii, B. henselae (Houston-1), B. henselae (Marseille), B. quintana, and B. vinsonii subsp. vinsonii. An overall Bartonella spp. seroprevalence of 16.1% was found, with a predominance of immunoreactivity to B. elizabethae, at 14.1%; B. grahamii, 2.6%; B. henselae (Houston-1), 1.2%; B. henselae (Marseille), 1.8%; B. quintana, 0.2%; and B. vinsonii subsp. vinsonii, 0.0%. Univariate and multivariate analyses of epidemiological and demographical information revealed an increased rate of B. elizabethae seropositivity in blood donors working outdoors, being out in the wild a minimum of once a week, hunting moose, having cat contact, and travelling to Eastern Europe. Living in the southern region of Sweden (Lund area) was associated with decreased seropositivity to B. elizabethae.