A five-laboratory collaborative study was undertaken to determine the precision and accuracy of broth microdilution susceptibility tests of Streptococcus pneumoniae isolates performed with Haemophilus test medium (HTM) compared with tests performed with lysed horse blood-supplemented Mueller-Hinton broth (LHB). The intra-and interlaboratory reproducibilities of MICs of 10 antimicrobial agents determined with the two media were found to be quite similar and highly reproducible in both media. On the basis of favorable performance in this study, S. pneumoniae ATCC 49619 is recommended as a quality control strain to assess the performance of HTM when this medium is used for testing of pneumococci. Testing of 293 unique clinical isolates of S. pneumoniae with both media in the respective participant laboratories allowed a direct comparison of MIC results and a calculation of interpretive error rates. Although there were some slight differences between MICs determined with HTM and MICs determined with LHB, few very major or major errors resulted from testing the clinical isolates against the 10 antimicrobial agents. However, MIC-interpretive criteria specific for S. pneumoniae should be developed and promulgated through a national consensus mechanism.