The LEADER Program (2007) monitors for emerging linezolid resistance in sampled US medical centers, initiated in 2004. For the current reported year, the number of sites participating was increased from 50 to 60 institutions representing all 9 US census regions with 100 target organisms per site (6305 isolates, 105.1% compliance to protocol design). The organisms tested by reference broth microdilution methods were Staphylococcus aureus (3318), coagulase negative staphylococci (CoNS, 1020), enterococci (705), Streptococcus pneumoniae (622), and viridans group (249) or beta-hemolytic streptococci (391); also, D-test was used to determine inducible clindamycin resistance in S. aureus strains. Linezolid remained very potent against all sampled species with MIC(90) results ranging from 1 microg/mL (streptococci and CoNS) to 2 microg/mL (S. aureus and enterococci). Only 0.44% of sampled strains were nonsusceptible to linezolid, compared with 0.45% in 2006. The nonsusceptible strains (23) were usually staphylococci (20) or Enterococcus faecium (8), each with defined target mutations (G2576T, 24 strains) or a novel mobile cfr element in staphylococci (2 strains). In conclusion, linezolid activity sampled by the 4th year of this LEADER Program showed sustained potency and spectrum (99.56% susceptibility). Although the nonsusceptible strain isolation rates remained stable, a new plasmid-mediated ribosomal-based resistance mechanism emerged in S. aureus and Staphylococcus epidermidis strains from Arizona and Ohio. The LEADER Program appears to be an effective and sensitive surveillance tool to detect novel resistance phenotypes and genotypes.