The molecular mechanisms contributing to high-level erythromycin resistance in Campylobacter jejuni and Campylobacter coli isolates were investigated. The A2075G mutation in the 23S rRNA target genes was identified in all high-level erythromycin-resistant isolates. A number of amino acid substitutions together with insertions and deletions were identified in the corresponding genes encoding L4 and L22 ribosomal proteins both of resistant and susceptible isolates. Amino acid substitutions identified in the resistant strains were located outside regions known to be altered in these proteins. The efflux pump inhibitor L-phenylalanine-L-arginine-beta-naphthylamide (PAbetaN) increased the susceptibility to erythromycin in one of four isolates displaying high-level erythromycin resistance, and reduced the minimal inhibitory concentration displayed by an erythromycin-susceptible C. coli isolate. The A2075G mutation in the 23S rRNA appeared to be the main contributor to high-level erythromycin resistance in Campylobacter. Other mutations/amino acid substitutions found in the 50S ribosomal subunit encoding proteins L4 and L22 do not appear to be linked to the high-level erythromycin-resistant phenotype. Active efflux contributes to the intrinsic resistance to erythromycin in Campylobacter and may contribute to high-level resistance in some isolates.