The reproducibility of identification and biochemical reactions for five different reference organisms of Enterobacteriaceae; Proteus vulgaris, Klebsiella pneumoniae, Escherichia coli, Serratia marcescens, and Enterobacter cloacae, were evaluated using the updated MS-2 system software (Abbott Laboratories, Diagnostic Division, Irving, Tex.) in a collaborative study involving 11 laboratories. When a total of 220 randomly coded test organisms were blindly examined, the MS-2 system correctly identified 92.7 and 86.8% for over 80 and 90% probability identification, respectively. Four organisms, P. vulgaris, K. pneumoniae, E. coli, and S. marcescens, were correctly identified in all laboratories with high probability, but 9 of 44 tests of Enterobacter cloacae resulted in misidentifications or low-likelihood (less than 80%) identifications. Accuracy was directly related to level of experience and familiarity with the MS-2 system in the individual laboratories. Biochemical reactions varied among the identification trials, especially in the identification of S. marcescens and Enterobacter cloacae. Among a total of 44 subcultures for each organism, 10 different biochemical patterns for P. vulgaris, 6 for K. pneumoniae, 9 for E. coli, 15 for S. marcescens, and 14 for Enterobacter cloacae were obtained. The results indicate that the MS-2 system performs with high accuracy and reproducibility in identifying Enterobacteriaceae, except for Enterobacter cloacae.