Three Gram‐negative, rod‐shaped bacteria that were found intracellularly in two environmental and one clinical
Acanthamoeba
sp. isolates were analysed. Two endocytobionts showing a parasitic behaviour were propagated successfully outside their amoebal host cells and were identified subsequently by comparative 16S rRNA sequence analysis as being most closely affiliated with
Flavobacterium succinicans
(99% 16S rRNA sequence similarity) or
Flavobacterium johnsoniae
(98% 16S rRNA sequence similarity). One endocytobiont could neither be cultivated outside its original
Acanthamoeba
host (
Acanthamoeba
sp. TUMSJ‐321) nor transferred into other amoebae. Electron microscopy revealed that the amoebal trophozoites and cysts were almost completely filled with cells of this endosymbiont which are surrounded by a host‐derived membrane. According to 16S rRNA sequence analysis, this endosymbiont could also be assigned to the
Cytophaga
–
Flavobacterium
–
Bacteroides
(CFB) phylum, but was not closely affiliated to any recognized species within this phylogenetic group (less than 82% 16S rRNA sequence similarity). Identity and intracellular localization of this endosymbiont were confirmed by application of a specific fluorescently labelled 16S rRNA‐targeted probe. Based on these findings, we propose classification of this obligate
Acanthamoeba
endosymbiont as ‘
Candidatus
Amoebophilus asiaticus’. Comparative 18S rRNA sequence analysis of the host of ‘
Candidatus
Amoebophilus asiaticus’ revealed its membership with
Acanthamoeba
18S rDNA sequence type T4 that comprises the majority of all
Acanthamoeba
isolates.