Pseudomonas cepacia is a significant pathogen in children and young adults with cystic fibrosis, and prevention of its acquisition has become an important goal in patient management. Although it is now clear that this bacterium can be transmitted from person to person, the frequency of this mode of acquisition and the measures required to prevent it are controversial. In this report we describe the use of a novel genotyping method to extend our previous investigation of person to person transmission of P. cepacia among patients with cystic fibrosis attending an educational program. Three (20%) of 15 individuals acquired P. cepacia after contact with a chronically colonized patient. Analysis revealed that the isolates recovered from the three newly colonized patients were the same as that from the index patient. We also demonstrated that pulmonary colonization with P. cepacia may not be detected by currently recommended culture methods for as long as 2 years after acquisition. These data indicate a need to develop more sensitive means of detecting P. cepacia colonization in order better to understand host-pathogen interaction and to optimize preventive strategies.