With the occurrence of extended-spectrum beta-lactamases (ESBLs) in Pseudomonas aeruginosa being increasingly reported worldwide, there is a need for a reliable test to detect ESBLs in clinical isolates of P. aeruginosa. In our study, a total of 75 clinical isolates of P. aeruginosa were studied. Nitrocefin tests were performed to detect the beta-lactamase enzyme; isoelectric focusing electrophoresis, PCR, and PCR product sequencing were designed to further characterize the contained ESBLs. Various ESBL-screening methods were designed to compare the reliabilities of detecting ESBLs in clinical isolates of P. aeruginosa whose beta-lactamases were well characterized. Thirty-four of 36 multidrug-resistant P. aeruginosa clinical isolates were positive for ESBLs. bla(VEB-3) was the most prevalent ESBL gene in P. aeruginosa in our study. Among the total of 34 isolates that were considered ESBL producers, 20 strains were positive using conventional combined disk tests and 10 strains were positive using a conventional double-disk synergy test (DDST) with amoxicillin-clavulanate, expanded-spectrum cephalosporins, aztreonam, and cefepime. Modifications of the combined disk test and DDST, which consisted of shorter distances between disks (20 mm instead of 30 mm) and the use of three different plates that contained cloxacillin (200 microg/ml) alone, Phe-Arg beta-naphthylamide dihydrochloride (MC-207,110; 20 microg/ml) alone, and both cloxacillin (200 microg/ml) and MC-207,110 (20 microg/ml) increased the sensitivity of the tests to 78.8%, 91.18%, 85.29%, and 97.06%.