Cross-infection with Pseudomonas aeruginosa is an emerging issue in the care of patients with cystic fibrosis (CF). This study sought to determine the extent of, and patient factors associated with, cross-infection in a tertiary referral adult CF centre. P. aeruginosa isolates were genotyped into two groups between November 2001 and February 2003, using pulsed-field gel electrophoresis after DNA digestion by the SpeI endonuclease, and identified as clustered if there was >80% homology in the macrorestriction profiles. Patient factors and measures of disease severity were identified a priori. In total, 157 out of 227 patients had a P. aeruginosa isolate genotyped. Of these, 94 patients (60%) were infected with clustered genotypes and 47 (30%) were infected with the newly described "Midlands 1" (Md1) genotype. A further 18 patients were infected with the previously identified "Liverpool" genotype and two with the "Manchester" genotype. Logistic regression analysis revealed that the predominant predictor of infection with Md1 was age at the time of referral to the centre, suggesting that infection may have occurred prior to referral in some patients. Md1 demonstrated a relatively benign anti-biogram and did not appear to be associated with more severe disease. In conclusion, the present study provides further evidence of the emerging importance of Pseudomonas aeruginosa cross-infection in cystic fibrosis.