Although plasmid-mediated AmpC beta-lactamases were first reported in the late 1980s, many infectious disease personnel remain unaware of their clinical importance. These enzymes are typically produced by isolates of Escherichia coli, Klebsiella spp., Proteus mirabilis, and Salmonella spp. and are associated with multiple antibiotic resistance that leaves few therapeutic options. Plasmid-mediated AmpC beta-lactamases have been associated with false in vitro susceptibility to cephalosporins. Many laboratories do not test for this resistance mechanism because current tests are inconvenient, subjective, lack sensitivity and/or specificity, or require reagents that are not readily available. In this study a new test, the AmpC disk test, based on filter paper disks impregnated with EDTA, was found to be a highly sensitive, specific, and convenient means of detection of plasmid-mediated AmpC beta-lactamases in organisms lacking a chromosomally mediated AmpC beta-lactamase. Using cefoxitin insusceptibility as a screen, the test accurately distinguished AmpC and extended-spectrum beta-lactamase production and differentiated AmpCs from non-beta-lactamase mechanisms of cefoxitin insusceptibility, such as reduced outer membrane permeability. The test is a potentially useful diagnostic tool. It can provide important infection control information and help to ensure that infected patients receive appropriate antibiotic therapy.