Retapamulin, the first pleuromutilin antimicrobial agent approved for the topical treatment of skin infections in humans, was tested against 987 clinical isolates representing 30 species and/or resistance groups. MICs were determined along with disk diffusion zone diameters using a 2-g disk. Population distribution and MIC versus disk zone diameter scattergrams were analyzed to determine microbiological MIC cutoff values and inhibition zone correlates. Minimum bactericidal concentrations were performed on a smaller subset of key species. The retapamulin MIC 90 against 234 Staphylococcus aureus isolates and 110 coagulase-negative staphylococci was 0.12 g/ml. Retapamulin MIC 90 s ranged from 0.03 to 0.06 g/ml against beta-hemolytic streptococci including 102 Streptococcus pyogenes, 103 Streptococcus agalactiae, 59 group C Streptococcus, and 71 group G Streptococcus isolates. The MIC 90 against 55 viridans group streptococci was 0.25 g/ml. Retapamulin had very little activity against 151 gramnegative bacilli and most of the Enterococcus species tested. Based on the data from this study, for staphylococci, MICs of <0.5, 1, and >2 g/ml with corresponding disk diffusion values of >20 mm, 17 to 19 mm, and <16 mm can be proposed for susceptible, intermediate, and resistant microbiological cutoffs, respectively. For beta-hemolytic streptococci, a susceptible-only MIC of <0.25 g/ml with a corresponding disk diffusion value of >15 mm can be proposed for susceptible-only microbiological cutoffs.