We examined the penicillin and macrolide resistance of 496 strains of Streptococcus pneumoniae (S. pneumoniae) isolated at Showa University Hospital from November 2004 to May 2005. According to the classification established by the Clinical and Laboratory Standards Institute, the ratio of penicillin susceptible S. pneumoniae (PSSP) was 25.8%, penicillin intermediate S. pneumoniae was 35.9% and penicillin resistant S. pneumoniae (PRSP) was 38.3%. The ratios of macrolide resistant S. pneumoniae were 85.3% for erythromycin and 76.2% for clarithromycin. S. pneumoniae strains were isolated mainly from pediatric patients, and the ratios of PRSP were similar between outpatients (39.8%) and inpatients (45.6%). We screened for mutations in pbp1a, pbp2b and pbp2x, and the retention rate of the macrolide resistance genes, ermB and mefA in 90 strains isolated in the same period. Seventy two strains had at least one mutation in the pbp genes. Interestingly, some of the penicillin susceptible strains had one or two pbp mutations, suggesting a progressive genetic acquirement of penicillin resistance. In screening for retention of the macrolide resistance genes, we found that 42 strains(46.7%) had ermB, 19 strains (21.1%) had mefA and 13 strains (14.4%) had both ermB and mefA. The possession of resistance genes and the minimal inhibitory concentration indicate that the resistance to erythromycin and clarithromycin were induced by ermB or mefA, and the resistance to clindamycin was induced only by ermB. Among the 72 strains with pbps mutations, 65 strains (90.3%) had ermB or mefA or both. Together, these results show that the strains with pbp mutations were being selected and, after acquiring the macrolide resistance gene, transform to multidrug resistant S. pneumoniae.