Macrolide resistance in Streptococcus pneumoniae is usually caused by the presence of the erm(B) or mef(A) resistance determinants. The aim of the present study was to identify the predominant macrolide resistance mechanisms among erythromycin-resistant S. pneumoniae isolated in a university hospital, Ankara, Turkey. A total of 669 S. pneumoniae strains were isolated from clinical specimens of patients admitted to the hospital between 1994-2002. The minimum inhibitory concentrations (MICs) of penicillin G, erythromycin A and clindamycin were determined by the agar dilution method according to NCCLS guidelines. Ninety-one (13.6%) isolates were resistant to erythromycin. Erythromycin-resistant isolates were examined for their macrolide resistance phenotypes by a triple disc diffusion assay. It assigned 57 (62.6%) of the 91 erythromycin-resistant pneumococci to cMLS(B) phenotype, 19 (20.9%) to iMLS(B) phenotype and 15 (16.5%) to M phenotype. All erythromycin-resistant isolates were analyzed by PCR for the presence of erm(B) and mef(A) determinants. The isolates were characterized for the underlying resistance genotype, with 83.5% having erm(B), 16.5% having the mef(A) genotypes. This study provides further evidence of the dissemination of macrolide-resistant mutants in pneumococci as the use of new, long-acting macrolides increases. This is the first article about MLSB resistance phenotypes and genotypes of S. pneumoniae from Turkey and it emphasizes the need for future epidemiological monitoring of macrolide-resistant pneumococci.