Anaerobic microorganisms (364 gram-negatives and 154 gram-positives) were investigated with respect to their behaviour against aqueous solutions of potassium hydroxide as well as their sensitivity to polymyxin and vancomycin by means of the disk diffusion test. Whereas the gram-negative anaerobes showed a good correlation (97.5%) with a positive KOH test, only 88.3% of the gram-positive microorganisms exhibited a negative KOH reaction. Therefore, in cases of ambiguous gram-negative staining results, the KOH test alone cannot give sufficient informations about the exact gram reaction. It was shown, however, that together with the determination of resistance to polymyxin and vancomycin, this uncertainty remains only for microorganisms showing a positive KOH reaction, polymyxin resistance, and vancomycin sensitivity. Therefore, in combination with the polymyxin and vancomycin disk diffusion test, the KOH test is recommended as an additional means to determine the gram reaction of anaerobes.