The emergence of macrolide- and lincosamide-resistant Streptococcus pneumoniae is a worldwide concern. Of particular interest is the increasing prevalence of erythromycin and clindamycin-resistant isolates containing both erm(B) and mef genes. This study determined the prevalence of erythromycin and clindamycin resistance in 596 clinical S. pneumoniae isolates from 2 adult tertiary care hospitals over a 4-year period (2001-2004). Erythromycin resistance increased from 24% to 34%, but S. pneumoniae isolates resistant to clindamycin as well as to erythromycin increased from 3% in 2001 to 15.5% in 2004 (5-fold increase). Among erythromycin-resistant isolates, those also resistant to clindamycin (MLS(B) phenotype) increased 3-fold (12.8-45%). Of forty-one erythromycin/clindamycin-resistant S. pneumoniae isolates tested, 29 (71%) contained both erm(B) and mef(E) genes. Pulsed-field gel electrophoresis performed on 28 erm(B) + mef(E) positive isolates identified 2 predominant and possibly related clones, which made up 64% of the isolates.