Objectives: The aim of this study was to investigate the persistence of Campylobacter species, strain types, antibiotic resistance and mechanisms of tetracycline resistance in poultry flocks treated with chlortetracycline. Methods: Three commercially reared broiler flocks, naturally colonized with Campylobacter, were treated with chlortetracycline under experimental conditions. The numbers of Campylobacter isolated, and the species, flaA short variable region allele, and antimicrobial resistance of isolates were determined. Results: For two of three flocks, tetracycline-resistant strains predominated prior to chlortetracycline exposure. Presence of the antibiotic had no discernible effect on the numbers or types of Campylobacter and the tetracycline-resistant strains persisted in numbers similar to those observed before treatment. With all flocks, some faecal samples were obtained that contained no Campylobacter, irrespective of exposure to chlortetracycline; this was more common as the birds grew older. For the third flock, tetracycline-resistant Campylobacter were in the minority of samples before and during exposure to chlortetracycline, but at sampling times after this, no resistant strains were found in the treated (or untreated) birds, irrespective of exposure to the antibiotic. All tetracycline-resistant isolates (MICs 16 to > 128 mg/L) contained tet (O) and, for some isolates, this was transferable to Campylobacter jejuni 81116. The efflux pump inhibitor PAβN reduced the MICs of tetracycline for these isolates by 4-fold, suggesting that an intact efflux pump, presumably CmeABC, is required for high-level tetracycline resistance. Conclusions: Our data indicate that chlortetracycline treatment does not eradicate tetracycline-resistant Campylobacter spp. from poultry. However, if a low number of resistant isolates are present, then the antibiotic pressure appears insufficient to select such strains as the dominant population. © The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.