The efficacy of vaccinating poultry against histomonosis was demonstrated recently. In the present study, the reversion to virulence and the residual pathogenicity of an in vitro attenuated, clonal strain of Histomonas meleagridis were tested in two consecutive experiments. The European Pharmcopoeia (Ph. Eur.) monograph for testing such features for coccidiosis live vaccines in chickens has served as a guideline. In the first experiment, attenuated histomonads were used in successive infection cycles in five groups of either five chickens or turkeys, respectively. All birds were killed at 14 days post infection (d.p.i.) to record lesion scores (LS) from livers and caeca. In the second experiment, the 5 times in vivo passaged histomonads were used to infect groups of 30 chickens and turkeys each, together with birds infected with virulent H. meleagridis. At three different time points 10 birds/group were killed and tissues of caeca, livers and lungs were used for PCR and immunohistochemistry (IHC) to confirm the presence of parasites. In the first experiment, various lesion scores were recordable in the livers and caeca of turkeys, with the highest LS 4 noticed once in the liver. In comparison, no lesions were seen in organs from chickens. In the second experiment, only mild lesions in the caeca of both turkeys and chickens were found. Liver lesions recorded as score 1 were noticed in just one individual of each species. PCR and IHC revealed that the attenuated and backpassaged histomonads were not present in liver samples but confined to the caeca, different to virulent H. meleagridis. Overall, no clinical signs or death occurred in turkeys or chickens inoculated orally and cloacally with 10(4) backpassaged histomonads in comparison with virulent parasites. Consequently, for the first time, the stable attenuation and safety of histomonads has been demonstrated, thus providing major implications for vaccine development.