Adult ticks were collected in a rural area of central Greece in order to isolate and identify rickettsiae. A hemolymph test using Gimenez staining was used for detection, while simultaneous isolation was performed using the shell-vial technique. Serologic, antigenic, and genomic characterization of the isolates was achieved by microimmunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP), and pulsed-field gel electrophoresis (PFGE), respectively. Although none of the 242 collected ticks was positive by the hemolymph test, one rickettsial isolate, designated GS, was obtained by the shell-vial technique. This isolate originated from a female Rhipicephalus sanguineus. Microimmunofluorescence serologic typing by the method of Philip and others demonstrated that GS belongs to the same serotype as the recently isolated Rickettsia massiliae (Mtul). Protein analysis by SDS-PAGE and immunoblotting by Western blot revealed similar profiles between the two rickettsiae. Using Alu I, Rsa I, and Pst I restriction endonucleases in PCR-RFLP analysis, GS and R. massiliae were found to possess identical restriction sites. However, PFGE showed differences when the two genomes were digested with Bss HII and Sma I restriction endonucleases, in spite of their equal size. In conclusion, the first rickettsial isolation in Greece was found to be antigenically identical and genotypically close to the French isolate R. massiliae, despite small differences showed by PFGE.