To assess chlorine susceptibility of Legionella pneumophila grown from two amoebic hosts, Acanthamoeba castellanii and Hartmannella vermiformis. After being released from amoebae, Leg. pneumophila were chlorinated at 2 and 5 mg l(-1) for 5 min-24 h. Bacterial culturability and cytoplasmic membrane deterioration were quantified by culture assay on BCYE alpha agar and BacLight stains coupled with a fluorescent microscope, respectively. Chlorination reduced the culturability of Leg. pneumophila by 2.93-4.59 log CFU ml(-1) and damaged cellular membrane by 53.8-99.2%. Moreover, cells released from H. vermiformis exhibited significantly lower degrees in culturability reduction (P = 0.0008) and membrane deterioration (P < 0.0001) when compared with those from A. castellanii. The amoebic genus is the most significant parameter affecting cytoplasmic membrane integrity of chlorinated Legionella (P < 0.0001), followed by free chlorine concentration (P = 0.042). Legionella pneumophila replicated from H. vermiformis possess greater chlorine resistance than the cells from A. castellanii. This study shows the heterogeneity of amoebae-grown Leg. pneumophila in chlorine susceptibility, which should be considered in the control of legionellae proliferation, particularly in the systems where H. vermiformis is dominant, e.g. hot water plumbing.