In this study, we present a novel method to isolate and enrich low concentrations of Campylobacter pathogens. This method, Acanthamoeba-Campylobacter coculture (ACC), is based on the intracellular survival and multiplication of Campylobacter species in the free-living protozoan Acanthamoeba polyphaga. Four of the Campylobacter species relevant to humans and livestock, Campylobacter jejuni, C. coli, C. lari, and C. hyointestinalis, were effectively enriched by the coculture method, with growth rates comparable to those observed in other Campylobacter enrichment media. Studying six strains of C. jejuni isolated from different sources, we found that all of the strains could be enriched from an inoculum of fewer than 10 bacteria. The sensitivity of the ACC method was not negatively affected by the use of Campylobacter-selective antibiotics in the culture medium, but these were effective in suppressing the growth of seven different bacterial species added at a concentration of 10(4) CFU/ml of each species as deliberate contamination. The ACC method has advantages over other enrichment methods as it is not dependent on a microaerobic milieu and does not require the use of blood or other oxygen-quenching agents. Our study found the ACC method to be a promising tool for the enrichment of Campylobacter species, particularly from water samples with low bacterial concentrations.