Since plasmid-mediated metallo-beta-lactamase (MBL)-producing Pseudimonas aeruginosa was reported in Japan, MBL-producing Gram-negative rods (GNRs) have emerged worldwide. We developed a way to detect MBL-producing GNRs in routine examination using broth microdilution with the MBL inhibitor sodium mercaptoacetate (SMA) in frozen plates. Between 1996 and 2005, we evaluated this and other methods, including broth microdilution with another MBL inhibitor dipicolinic acid (DPA) in dry plates, conventional PCR, and a combined simple DNA preparation and enzymatic PCR product detection. The combined method is suitable for detecting IMP-type MBL-producing GNRs from numerous isolates. Broth microdilution with SMA at a concentration of 400 microg/mL had high performance and detected most PCR-positive MBL-producing GNRs in routine antimicrobial susceptibility testing. DPA in dry plates at 400 microg/mL yielded false positive results in 11.4% of isolates but worked satisfactorily at 175 microg/mL and 400 microg/mL of SMA in frozen plates. Until 1996, MBL had been detected from only 6 bacterial species, i.e., Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas stutzeri, Achromobacter xylosoxidans, Serratia marcescens, and Citrobacter freundii, MBL-producing GNRs were later found in other glucose nonfermenting GNRs such as Acinetobacter baumanii, Acinetobacter lwoffii, and Burkholderia cepacia complex and Enterobacteriaceae. Most MBL-producing bacteria were multidrug resistant and no antimicrobial agents exist that are active against such isolates in monotherapy, making their rapid detection very important in controlling infection control.