Gram-positive cocci are important causes of both nosocomial and community-acquired infections, and antimicrobial resistance among these pathogens has become an important problem worldwide. Since resistance among these organisms can vary substantially by geographic location, we conducted a multicenter surveillance study with isolates from five Latin American countries (15 medical centers). Quinupristin/dalfopristin (formerly RP-59500) is a novel streptogramin combination with focused activity against Gram-positive cocci, many exhibiting emerging resistance. The in vitro activity of quinupristin/dalfopristin and 12 other antimicrobial agents were evaluated against 1,948 strains including Staphylococcus aureus (747 strains), coagulase-negative staphylococci (CoNS;446 strains), enterococci (429 strains), and various Streptococcus spp. (326 strains). Oxacillin resistance was observed in 41% of S. aureus (MIC, <e; 2 microg/ml or >e; 13 mm) and 40% of CoNS (MIC, <e; 0.25 microg/ml or >e; 18 mm). Vancomycin, teicoplanin, and quinupristin/dalfopristin (MIC(90), 0.25 - 1 mg/ml) remained effective against all strains, but cross-resistance was high among other tested drugs. The quinupristin/dalfopristin MIC(50) for Streptococcus pneumoniae and other streptococci was only 0.5 mg/ml (13% to 28% were penicillin-resistant; 12% to 22% were macrolide-resistant). Enterococci demonstrated variable inhibition by quinupristin/dalfopristin depending upon identification and the susceptibility testing method used. The demonstrated quinupristin/dalfopristin activity against Enterococcus faecium was confirmed, but potential species identification errors with various commercial systems continue to confuse susceptibility statistics, even though some strains of E. faecium confirmed by PCR-based or other molecular identification techniques did have quinupristin/dalfopristin MICs of >e; 4 microg/ml. Most important, glycopeptide-resistant enterococci are rapidly emerging in Latin America, and quinupristin/dalfopristin appears active against many of these isolates as well as having potency against nearly all staphylococci and streptococci tested at <e; 2 microg/ml or having a zone diameter of >e; 16 mm. Comparisons to GSMART results from other continents show nearly identical quinupristin/dalfopristin activity for each Gram-positive species tested. These results define the role of quinupristin/dalfopristin in Latin American medical centers and provide a benchmark for future in vitro comparisons.