The in vitro activity of GAR-936, a new semisynthetic glycylcycline, was evaluated in comparison with two tetracyclines and the disk diffusion susceptibility test was assessed. Nearly 700 recent clinical isolates were tested by reference broth microdilution and disk diffusion (two disk contents) methods. Among the Enterobacteriaceae, GAR-936 was generally two- to 16-fold more active than other tetracyclines. All enteric bacilli MIC90 results were < or = 4 micrograms/mL; the exception being Proteus mirabilis and indole-positive Proteae (> or = 8 micrograms/mL). GAR-936 demonstrated excellent activity against all Gram-positive cocci with Enterococcus spp. (including vancomycin-resistant isolates) inhibited at 0.25 microgram/mL (GAR-936 MIC90, 0.12 or 0.25 microgram/mL) and the oxacillin-resistant Staphylococcus aureus strains were inhibited at < or = 0.25 microgram/mL. GAR-936 demonstrated good potency against several non-fermentative bacteria, but possessed limited activity against Pseudomonas aeruginosa (MIC50, 8 micrograms/mL). In vitro susceptibility test methods were developed (disk diffusion versus reference MIC results) and tentative breakpoints were proposed. Using susceptibility criteria of either < or = 2 or < or = 4 micrograms/mL, GAR-936 in vitro susceptibility tests demonstrated rare significant serious inter-method discords (< or = 1.2%) and an absolute agreement between test results of 92.3 to 96.2%. These results indicate that GAR-936 has potent in vitro activity against a wide range of clinically important rapidly growing pathogenic bacteria, and that this novel glycylcycline candidate for clinical use should be further developed.