ABSTRACTFollowing cultivation-dependent and -independent techniques, we investigated the microbiota associated withBactrocera oleae, one of the major agricultural pests in olive-producing countries. Bacterial 16S rRNA gene libraries and ultrastructural analyses revealed the presence of several bacterial taxa associated with this insect, among whichAcetobacter tropicaliswas predominant. The recent increased detection of acetic acid bacteria as symbionts of other insect model organisms, such asAnopheles stephensi(G. Favia et al., Proc. Natl. Acad. Sci. USA 104:9047-9051, 2007) orDrosophila melanogaster(C. R. Cox and M. S. Gilmore, Infect. Immun. 75:1565-1576, 2007), prompted us to investigate the association established betweenA. tropicalisandB. oleae. Using anA. tropicalis-specific PCR assay, the symbiont was detected in all insects tested originating from laboratory stocks or field-collected from different locations in Greece. This acetic acid bacterium was successfully established in cell-free medium, and typing analyses, carried out on a collection of isolates, revealed that differentA. tropicalisstrains are present in fly populations. The capability to colonize and lodge in the digestive system of both larvae and adults and in Malpighian tubules of adults was demonstrated by using a strain labeled with a green fluorescent protein.