Detecting resistance in gram-negative microorganisms has a strong clinical and epidemiological impact, but there is still a great deal of debate about the most sensitive phenotypic method and whether in vitro susceptibility results should be interpreted. The present work reviews the phenotypes and mechanisms of resistance to beta-lactams, quinolones and aminoglycosides in gram-negative bacilli and also revises the different phenotypic methods used for their detection. A clinical interpretation of in vitro susceptibility results is also discussed. Extended-spectrum and inhibitor resistant beta-lactamases, AmpC type beta-lactamases and carbapenemases are thoroughly reviewed. As regards quinolones, the resistance mediated both by plasmids and by mutations in the DNA gyrase and the topoisomerase IV genes is also reviewed. This report includes resistance patterns to aminoglycosides caused by modifying enzymes. Phenotypic detection of beta-lactam resistance in Neisseria spp. and Haemophilus influenzae is also reviewed in a separate section.