A four-center study in which a total of 1,082 recent clinical isolates of members of the family Enterobacteriaceae and Pseudomonas aeruginosa were examined versus 11 antimicrobial agents with the bioMérieux Vitek susceptibility test system (Hazelwood, Mo.) and the GNS-F6 card was conducted. In addition, a challenge set consisting of the same 200 organisms was examined in each of the four participating laboratories. Results obtained with the Vitek system were compared to MICs determined by a standardized broth microdilution method. For purposes of comparison, susceptibility categories (susceptible, intermediate, or resistant) were assigned on the basis of the results of both methods. The result of the broth microdilution test was considered definitive. The total category error rate with the Vitek system and the recent clinical isolates (11,902 organism-antimicrobial comparisons) was 4.5%, i.e., 1.7% very major errors, 0.9% major errors, and 1.9% minor errors. The total category error rate calculated from tests performed with the challenge set (i.e., 8,800 organism-antimicrobial comparisons) was 5.9%, i.e., 2.2% very major errors, 1.1% major errors, and 2.6% minor errors. Very major error rates higher than the totals were noted with Enterobacter cloacae versus ampicillin-sulbactam, aztreonam, ticarcillin, and ticarcillin-clavulanate and with P. aeruginosa versus mezlocillin, ticarcillin, and ticarcillin-clavulanate. Major error rates higher than the averages were observed with Proteus mirabilis versus imipenem and with Klebsiella pneumoniae versus ofloxacin. Excellent overall interlaboratory reproducibility was observed with the Vitek system. The importance of inoculum size as a primary determinant in the accuracy of susceptibility test results with the Vitek system was clearly demonstrated in this study. Specifically, when an inoculum density fourfold higher than that recommended by the manufacturer was used, high rates of false resistance results were obtained with cell wall-active antimicrobial agents versus both the Enterobacteriaceae and P. aeruginosa.