Two commercially available immunofluorescence assays (IFA) were compared using historical sera evaluated for rickettsial antibodies by the Weil-Felix test. An IFA test produced by Focus Diagnostics prepared with Rickettsia rickettsii and R. typhi antigens was compared with a custom made kit from Fuller Laboratories with R. rickettsii, R. typhi, R. conorii and R. helvetica as antigens. The serum panel used for the comparison included Weil-Felix-positive and -negative samples. Cross-reactions were analyzed using serum samples from patients with clinical symptoms similar to those of rickettsiosis. When analyzing the data using the manufacturers' cut-off values, 41% of samples from presumably healthy blood donors were found positive for spotted fever group Rickettsia antibodies. This does not correlate to the general picture of rickettsiosis in Denmark. Furthermore, sera with Coxiella burnetii antibodies were found to be cross-reacting in both tests. When applying cut-off values calculated on the 95% percentile on data from blood donor serum samples, there was no significant difference between the two kits. Moreover, when using the newly established cut-off, cross-reactions were eliminated.