Reactions obtained with Hugh-Leifson's oxidation-fermentation (OF) medium in 10-ml. amounts (one tube), without oil overlay, correlated well with those obtained with the standard OF test (two tubes) and with phenol red broth for the fermentative and the nonfermentative Gram-negative bacilli tested. A comparison of tubed OF medium prepared by three different manufacturers indicated a significant lack of standardization for this type of medium. The advantages of the one-tube OF test are discussed.