Objectives: To investigate the effect of subinhibitory concentrations of ciprofloxacin, streptomycin, trimethoprim, ampicillin and erythromycin on mutation frequency in Streptococcus pneumoniae. Methods: Frequency of mutation to rifampicin resistance was determined in three clinical isolates grown with or without antibiotic treatment. dinB was analysed using PCR and DNA sequence determination. Results: Subinhibitory levels of ciprofloxacin and streptomycin increased the frequency of mutation to rifampicin resistance between 2- and 5-fold for all three isolates, which is comparable to the increase seen in mismatch repair mutants of this species. These increases appeared not to be dependent on the function of the error-prone DNA polymerase encoded by dinB, since one of the isolates was a naturally occurring deletion mutant for this gene. Trimethoprim increased the mutation frequency for two isolates, but not the dinB mutant; ampicillin and erythromycin had no significant effect on mutation frequencies for any isolate. Conclusions: Exposure to quinolones and aminoglycosides at subinhibitory concentrations may result in increased mutability in pneumococci, as well as selecting for resistance per se.