SCH27899 is an oligosaccharide, everninomicin antibiotic with activity primarily against Gram-positive pathogens. The activity of SCH27899 was evaluated against 360 routine clinical isolates by the broth microdilution (BMD), agar dilution (AD), disk diffusion (DD), and Etest (AB BIODISK, Solna, Sweden) methods. In addition, results from a nine center SCH27899 quality control (QC) trial were used to establish QC ranges. SCH27899 MICs for 330 Gram-Positive strains, including multiply-resistant staphylococci and enterococci, ranged from 0.015 to 1 microgram/ml with MIC90s of 0.12 to 0.5 microgram/ ml. SCH27899 had no measurable activity against the 30 selected Gram-negative strains tested (MICs, > 256 micrograms/ml), with the exception of Moraxella catarrhalis MICs, 0.12 microgram/ ml). Etest MICs for SCH27899 correlated well with AD and BMD results with > 90% of MICs within +/- one log2 dilutions of the reference test results. Three disk concentrations (2.5-, 5-, 10-microgram) of SCH27899 were evaluated, but minimal difference of zone diameters between disk drug contents was observed (+/- 2 mm). SCH27899 disk zone diameters correlated poorly with reference MICs due to small zone diameters (range, 11 to 22 mm) attributed to poor diffusion through agar mediums, a product of this compound's high molecular weight and solubility. The use of the DD method for SCH27899 was not recommended. The proposed MIC quality assurance limits for SCH27899 using Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 was 0.06 to 0.25 microgram/ml for both QC strains and methods. SCH27899 appears to be a eveminomicin-derivative widely active against important Gram-positive cocci, and in vitro dilution testing methods would be preferred for clinical use, validated by the recommended MIC control ranges cited in this report.