The mechanism of macrolide resistance was examined in 73 clinical isolates of Streptococcus pneumoniae. Two distinct resistance phenotypes were observed: high-level macrolides-lincosamides-streptogramin B (MLS) resistance and low-level macrolide resistance with lincosamide susceptibility. High-level MLS resistance was associated with the presence of ermAM. Strains with the low-level resistant phenotype (novel) were negative for ermA, ermC, ermAM, ereA, ereB and msrA by polymerase chain reaction (PCR) amplification with gene-specific primers. Ribosomes isolated from novel strains bound the same amount of [14C]-erythromycin as ribosomes from sensitive strains. These novel strains also did not inactivate the macrolide. The novel mechanism was found in 41% of the erythromycin resistant S. pneumoniae examined.