Viridans group streptococci (VGS) are commonly isolated from the blood of hospitalized patients. The E test represents a convenient method for determining the MICs for VGS, but for this purpose it has not been well validated against reference methods. In this study, 180 unselected VGS isolates were identified to a species level, and the MICs of penicillin, cefuroxime, cefotaxime, and vancomycin were determined by both agar dilution and the E test. Available data regarding demographic and laboratory variables for each VGS bacteremic episode were collected, the significance of each VGS isolate was assessed, and the associations between and among laboratory and clinical variables were investigated. Among all VGS isolates, 68.3% (median of three runs) were found to be fully susceptible to penicillin by agar dilution. The E test and agar dilution showed average agreements (within ±1 dilution) of 92.2% for penicillin, 95.7% for cefuroxime 91.3% for cefotaxime, and 86.7% for vancomycin. Agreements over serial E tests and serial agar dilutions were excellent for -lactam agents (intraclass correlation coefficients, >0.9) but less impressive for vancomycin. Very major error rates for the E test were 0.7%, and combined major and minor error rates were within acceptable limits for all antimicrobial agents tested. Lysis-centrifugation culture methods were more often associated with clinically insignificant VGS isolates; otherwise, no associations between clinical and laboratory variables were noted.