Streptococcus pneumoniae is the micro-organism most frequently isolated from acute otitis media (AOM). Recently, drug-resistant or insensitive strains have been prevalent among pneumococcal AOM cases and were reportedly associated with persistent or recurrent otitis media. The purpose of this study was to examine the alterations of penicillin-binding proteins (PBPs) in Streptococcus pneumoniae isolated from AOM. The PBP samples from 21 clinically isolated strains were examined by 14C-penicillin binding assay and also by adding CCL and CDTR as competitors of 14C-penicillin. Reduced signals in PBP1A and PBP2X/2A were typically observed with the penicillin-resistant Streptococcus pneumoniae samples, while widely different PBP profiles were obtained in each strain of penicillin-insensitive Streptococcus pneumoniae. The competitive binding assay with penicillin-insensitive strains revealed the reduced affinity of CCL to PBPs in comparison with that of CDTR. The results of the penicillin-binding assay were consistent with the results from minimal inhibitory concentration analysis, and its potential usefulness for the prediction of drug resistance was demonstrated.