The aim of this study was to investigate whether Burkholderia cepacia is capable of survival and growth within the free-living amoeba Acanthamoeba polyphaga using a differential immunofluorescence assay of bacterial-amoebal cocultures and viable counts of bacteria determined after amoebal lysis. The numbers of intra-amoebal bacteria and the numbers of infected amoebae increased over time; although, when heat-killed bacteria were used, no intracellular bacteria were observed. These findings should be taken into account in future studies of environmental reservoirs of Burkholderia cepacia.