Sampling of the lower airways of the adult cystic fibrosis (CF) lung has received insufficient detailed consideration, with the importance of sampling strategies for bacteriological outcome not known. Although spontaneously expectorated sputum (SES) samples are often used for diagnostic bacteriological analysis, induced sputum (IS) methods have advantages. This study examined whether significant differences in bacterial content were detected when using a culture-independent, molecular profiling technique to analyze SES or IS samples. Moreover, this work examined what trends relating to bacterial species distributions and reproducibility were found in sequentially induced sputum samples and what implications this has for pathogen detection. Terminal restriction fragment length polymorphism (T-RFLP) analysis was performed on a SES sample and 4 subsequent IS samples taken at 5-min intervals from 10 clinically stable, adult CF patients. This was repeated over 3 sampling days, with variability between samples, induction periods, and sampling days determined. A diverse range of bacterial species, including potentially novel pathogens, was found. No significant difference in bacterial content was observed for either SES or serial IS samples. On average, the analysis of a single sample from any time point resolved ̃58% of total bacterial diversity achieved by analysis of an SES sample and 4 subsequent IS samples. The reliance on analysis of a single respiratory sample was not sufficient for the detection of recognized CF pathogens in all instances. Close correlation between T-RFLP and microbiological data in the detection of key species indicates the importance of these findings in routine diagnostics for the detection of recognized and novel CF pathogens.