Recent seroepidemiological studies and examinations of Ixodes ricinus ticks in Europe have demonstrated the presence of an emerging tick-borne infection with Rickettsia helvetica. We conducted a serosurvey in 168 Danish patients seropositive for borreliosis reflecting their exposure to I. ricinus ticks. A total of 21 patients (12.5%) had positive antibody titres to R. helvetica including 4 cases of seroconversion. None of the samples were positive for antibodies to Ehrlichia. We conclude that in humans exposed to I. ricinus ticks in Denmark the risk of acquiring rickettsial infection is for the first time demonstrated. In the same region of Denmark we collected 570 I. ricinus ticks from various sources, and examinations by PCR for Rickettsia were performed. Positive reactions were obtained in 23 ticks (4%), and R. helvetica was identified in all 13 of those for which sequencing was performed.