A total of 132 Streptococcus pneumoniae isolates collected between 2005 and 2006 in Japan were examined for susceptibility to telithromycin (TEL) and macrolide. The overall resistance to macrolide was 80%. Among the isolates, 128 strains had low-level TEL susceptibility (minimal inhibitory concentrations [MICs] 0.03-1 microg mL(-1)), suggesting that pneumococci with reduced susceptibility to TEL have appeared without prior exposure to the drug, although none of the isolates were assigned as TEL-resistant (breakpoint, > or = 4 microg mL(-1)). Eight of these isolates (MIC 0.5-1 microg mL(-1)) were analyzed for macrolide resistance determinants and genetic relatedness. They all carried mefE-mel, which encodes the macrolide efflux genetic assembly, and three also harbored ermB, which encodes rRNA methylase. Allele replacement mutagenesis of the corresponding genes in the clinical isolates revealed that reduced TEL susceptibility (MIC 1 microg mL(-1)) in S. pneumoniae may be caused by acquisition of the mefE-mel element only and additionally conferred by the ermB determinant.