The aims of this study were to evaluate the frequency of Achromobacter xylosoxidans infection in a cohort of cystic fibrosis patients, to investigate antimicrobial sensitivity, to establish possible clonal likeness among strains, and to address the clinical impact of this infection or colonization on the general outcome of these patients. The study was undertaken between January 2004 and December 2008 on 300 patients receiving care at the Regional Cystic Fibrosis Center of the Naples University "Federico II". Sputum samples were checked for bacterial identification. For DNA fingerprinting, pulsed-field gel electrophoresis (PFGE) was carried out. Fifty-three patients (17.6%) had at least one positive culture for A. xylosoxidans; of these, 6/53 (11.3%) patients were defined as chronically infected and all were co-colonized by Pseudomonas aeruginosa. Of the patients, 18.8% persistently carried multidrug-resistant isolates. Macrorestriction analysis showed the presence of seven major clusters. DNA fingerprinting also showed a genetic relationship among strains isolated from the same patients at different times. The results of DNA fingerprinting indicate evidence of bacterial clonal likeness among the enrolled infected patients. We found no significant differences in the forced expiratory volume in 1 s (FEV(1)) and body mass index (BMI) when comparing the case group of A. xylosoxidans chronically infected patients with the control group of P. aeruginosa chronically infected patients.