Gimenez staining of presumably axenic Acanthamoeba sp., strain HN-3, showed rod-shaped cytoplasmic inclusions. Electron microscopy of thin sections of the amebae showed these to be bacilli which measured 1.3 to 3.3 microns by 0.22 to 0.33 micron. Their cell envelopes were those typical of gram-negative bacteria, surrounded by an electron-translucent area that stains with ruthenium red, suggesting the presence of a capsule. The bacilli grew and reproduced in the cytoplasm of both trophozoites and cysts of Acanthamoeba sp. There was no evidence of a surrounding phagosomal or phagolysosomal membrane. They were retained by the ameba both during encystment and excystment. All attempts to isolate the endosymbionts in embryonated eggs and/or standard bacteriological media failed; and they persisted within the amebae for 1 to 6 mo despite temperature shocking or constant treatment of cultures with penicillin, streptomycin, chloramphenicol, tetracycline, erythromycin, polymyxin B, ampicillin, isoniazid, rifampicin, or gentamycin at concentrations of 10(-5) to 10(-3) M.