In an effort to identify bacteria that fail to give the expected Gram reaction, thus leading to misidentification, two nonstaining tests for Gram reaction, vancomycin and colimycin susceptibility testing and the Gram-Sure test (Remel, USA), were employed on 145 strains from 42 gram-negative and gram-positive genera with contradictory Gram stain results. The Gram-Sure test is a commercially available disk that detects the presence of L-alanine-aminopeptidase, an enzyme usually found only in the cell wall of gram-negative bacteria. In this test, aminopeptidase activity is detected using a substrate that can be hydrolyzed to produce a fluorescent compound under long-wave UV light. The commercial disk test and vancomycin plus colimycin susceptibility testing appeared to perform equally well except in the identification of Erysipelothrix and Lactobacillus, for which the commercial disk test was better, and Moraxella, for which vancomycin and colimycin susceptibility testing was more helpful. An advantage of the commercial disk test is that it can be performed in 10 min, whereas vancomycin and colimycin susceptibility testing requires at least 18 h. The commercial disk test is also less expensive than vancomycin and colimycin susceptibility testing. However, since the same results can be obtained with the 5 microg and 30 microg vancomycin disks, it is possible to use only one vancomycin disk, with the cost then being equivalent to that of the commercial disk test. The major inconvenience of the commercial disk test is the requirement of a UV ray. However, this test could be a useful tool for the identification of unusual organisms.