Simkania negevensis is a recently discovered intracellular organism that has been associated with respiratory tract infections. To determine the seroprevalence of the organism in adult Danes and to study the association between the organism and persistent cough, we developed an immunofluorescence assay based on S. negevensis infected Hep2 cells for antibody determination and a real time PCR assay for direct detection of the organism. Among 100 healthy blood donors, 41 (41%) had IgG antibodies to S. negevensis (cut-off titre =1:16) and the antibody level increased with increasing age (correlation coefficient 0.124, p=0.037). 80 of 185 patients (43%) with chronic cough had IgG antibodies to S. negevensis which was no different from the 41% in the control population (Chi2=0.13, p=0.72). None of the patients or controls had any detectable IgA antibodies to S. negevensis. PCR was performed on nasopharyngeal aspirates from a subgroup of 176 patients with persistent cough and in none of these was S. negevensis DNA detected. We conclude that the seroprevalence of S. negevensis is high in Denmark and positively correlated with age. However, we were unable to show an association between S. negevensis and persistent cough.