To evaluate the prevalence of spotted fever group rickettsiae along the Adriatic Coast of Croatia, 832 ticks were examined by hemolymph test, direct immunofluorescence, antigen-capture enzyme immunoassay, and polymerase chain reaction. Very good agreement was observed among direct immunofluorescence, polymerase chain reaction, and antigen-capture enzyme immunoassay. Twelve ticks that were positive by hemolymph test and negative by both direct immunofluorescence and polymerase chain reaction presumably do not represent spotted fever group rickettsiae. By direct immunofluorescence, spotted fever group rickettsiae were present in 12% of Rhipicephalus bursa, 10.6% of Rh. sanguineus, and 7.8% of Dermacentor marginatus. From the 98 ticks containing rickettsialike organisms by hemolymph test, seven spotted fever group rickettsial isolates were established in cell culture. Four isolates were identified as Rickettsia conorii. The antigencapture enzyme immunoassay, which utilizes a monoclonal antibody to antigens of the 135-kD surface protein shared among many members of the spotted fever group, is recommended for primary screening of tick samples because it is reliable and yet less labor-intensive than the hemolymph and direct immunofluorescence tests. Although the polymerase chain reaction is too expensive for use as a screening method, it is recommended for confirmation of positive screening results. In addition to the technologic advance of the antigen-capture enzyme immunoassay, this study documented by contemporary methods that R. conorii is present along the eastern Adriatic Coast not only in the classic vector, Rh. sanguineus, but also in Rh. bursa and D. marginatus.