Objectives: The first objective was to investigate the in vitro activity of telithromycin against respiratory tract pathogens in comparison with other antimicrobial agents. The second objective was to identify the influence of the erm(B) and mef(A) genes on the susceptibility of Streptococcus pneumoniae to telithromycin. Methods: The in vitro activity of telithromycin against S. pneumoniae, Moraxella catarrhalis and Haemophilus influenzae, isolated from the UK and 40 macrolide-resistant S. pneumoniae from four different countries was compared with a variety of antimicrobial agents. The 140 isolates were examined for the presence of the erm(B) and mef(A) genes. The impact of 5% CO2 on susceptibility testing was also investigated. Results: Telithromycin showed greatest activity against S. pneumoniae, but also had good activity against M. catarrhalis and H. influenzae, which was independent of their resistance profiles to other antibiotics. The MIC90 of telithromycin for S. pneumoniae was 0.12 mg/L, which was 64-fold lower than the lowest macrolide MIC; 21% of the S. pneumoniae were macrolide resistant. Thirty-eight per cent of the macrolide-resistant strains were erm(B)-positive and 62% were mef(A)-positive, but no strain contained both genes. The activity of telithromycin was similar to that of azithromycin against both M. catarrhalis and H. influenzae, Erythromycin was slightly less active: 1% and 8% of M. catarrhalis and H. influenzae, respectively, were resistant to erythromycin, but none were resistant to telithromycin. Five per cent of the S. pneumoniae strains and 4% of the H. influenzae strains changed from telithromycin susceptible to non-susceptible entirely because of the incubation conditions. The MIC50s and MIC90s of S. pneumoniae, M. catarrhalis and H. influenzae increased by one dilution when incubated in CO2. Conclusions: Telithromycin has shown high in vitro activity against S. pneumoniae, including those strains that are macrolide susceptible and resistant as well as M. catarrhalis and H. influenzae. This study has also demonstrated that there is no cross-resistance between erythromycin and telithromycin. The impact of 5% CO2 on susceptibility testing should be investigated further before providing definite guidelines on telithromycin susceptibility testing.