The aim of this study was to analyze the mechanisms of macrolide resistance in French clinical isolates of Streptococcus pneumoniae. A total of 838 strains of pneumococci were isolated in 1997 in Normandy, a region of western France, by 19 microbiology laboratories. Fifty-three percent had displayed diminished susceptibility to penicillin G and 50% were resistant to erythromycin. From this collection, 92 penicillin-intermediate or -resistant and 18 penicillin-susceptible strains resistant to erythromycin were studied. The presence of erm genes coding for ribosomal methylases and of mefE-like genes responsible for macrolide efflux was screened by a multiplex polymerase chain reaction and confirmed by DNA/DNA hybridization. Of the 110 strains studied, 108 were cross-resistant to erythromycin, spiramycin and clindamycin, including 105 strains containing ermB-related genes and three strains that contained a combination of ermB- and mefE-related genes. Two strains apparently susceptible to clindamycin but resistant to spiramycin also contained ermB-related genes. No strain was resistant to erythromycin alone or contained only a mef-like gene. Therefore, resistance to erythromycin is mostly related to ribosomal methylation in this region of France.