To investigate the prevalence of resistance to macrolide, lincosamide and streptogramin (MLS) antibiotics in Gram-positive cocci isolated in a Korean hospital, we tested the antibiotic susceptibility of 1097 clinical isolates of Staphylococcus aureus, coagulase-negative staphylococci (CNS) and enterococci to the macrolides erythromycin, clarithromycin, azithromycin and josamycin, the lincosamide clindamycin and the streptogramin pristinamycin. These three groups of organisms were mostly resistant to macrolides and lincosamide, but were commonly susceptible to pristinamycin. The resistance phenotypes of erythromycin-resistant isolates were determined by the double-disc test with erythromycin and clindamycin, which showed that most exhibited constitutive MLS resistance. In order to determine the prevalence of the resistance genotypes and the resistance mechanisms, the presence of the erm(A), erm(B), erm(C) and mef genes in the erythromycin-resistant isolates was identified by PCR analysis. The resistance was due mainly to the presence of erm(A) in S. aureus (82.5%), erm(B) in enterococci (55%) and erm(C) in CNS (47.2%).