We studied 198 macrolide-resistant S. pneumoniae isolates obtained from adults and children to evaluate whether 2-μg clindamycin disks can distinguish between isolates manifesting ermB- versus mefE-mediated resistance to clarithromycin and to determine the relative frequency with which each resistance mechanism occurred in these populations. The mefE gene was predominant among 109 isolates from children, occurring in 73.4% versus 50.6% of 89 isolates from adults. Three isolates (1.5%) did not amplify either gene. Among 125 mefE+ isolates, the MIC of clarithromycin at which 90% of the isolates tested were inhibited, determined by Etest, was 32 μg/ml versus >256 μg/ml in 70 ermB+ isolates. All ermB+ isolates were highly resistant to clindamycin (MICs >256 μg/ml), whereas all mefE+ isolates were susceptible to clindamycin using the 2-μg disk. Testing S. pneumoniae from the respiratory tract for susceptibility to clindamycin by agar disk diffusion is an easy and inexpensive method to estimate the frequency of resistance mediated by ermB in specific patient populations. Macrolide resistance mediated by ermB is usually of greater magnitude than that due to mefE. Clinical studies are needed to determine the significance of high- versus low-level macrolide resistance in S. pneumoniae.