We have been screening a cohort of 225 chromosomes from cystic fibrosis patients for mutations in the cystic fibrosis transmembrane regulator gene using a combination of DGGE,RNA-SSCP and DNA sequencing. A novel splice site mutation was detected by multiplex DGGE in a homozygous patient. Restriction-site generating PCR (RG-PCR) analysis demonstrated that both parents carried the same mutation. The molecular haplotype was the same. All the known ancestors came from the same (Veneto) region, and no consanguinity was documented up to the sixth generation.