Since 1972, there have been no reports of Mediterranean spotted fever (MSF) in Greece. In 1991, a seroepidemiologic survey was conducted in three rural villages in the Province of Fokida in central Greece, using both an immunofluorescence assay (IFA) and Western blot to evaluate the prevalence of specific IgG and IgM antibodies to Rickettsia conorii, and to compare these two techniques. Of 254 sera tested by IFA, 148 (58.3%) were positive for IgG at a titer > or = 32 and 117 (46.1%) at a titer > or = 64. Two sera were positive for IgM at a titer > or = 32. Among the IFA-positive sera, 115 reacted against the specific protein antigen (SPA) using Western blotting and were considered as confirmed positive results, indicating a seroprevalence of 45.3%. Using the SPA reaction as a reference, the IFA diagnostic value of a single serum is poor in such an endemic area, suggesting the need for more specific tests. The results of this study have led us to conclude that for seroepidemiologic use, the IFA is not specific for MSF. Thus, the IFA is useful for sero-epidemiologic analysis only in areas where the true prevalence of MSF has been estimated using Western blotting.