A coccoid endosymbiont was recovered from Hartmannella vermiformis OS101. The organism could be propagated in BGM and Vero cell cultures, was resistant to penicillin and exhibited structural characteristics comparable with Chlamydia spp. PCR using Chlamydia ompA gene and Coxiella-specific primers showed that the agent was distinct from chlamydiae or coxiellae. Determination of 16S ribosomal RNA signature sequences in combination with the morphological and developmental characteristics provided evidence that the isolate can be classified as Simkania negevensis or a closely related organism. Even the investigated signature sequences showed a high homology to those of the type strain of Simkania negevensis, its biological properties in amoebae cultures differed to that of strain Z.