Standard methods for determining the tuberculocidal activity of liquid chemical germicides are difficult to perform and require long incubation periods to obtain results. We report the development of a rapid, quantitative assay for tuberculocidal activity that uses bioluminescence to detect mycobacteria that survive exposure to these germicides. The test organism for this assay is a strain of Mycobacterium bovis BCG that contains a plasmid carrying the firefly luciferase gene fused to the BCG hsp60 promoter. The level of light produced, after the addition of the substrate luciferin, is proportional to the number of living bacteria present. In this assay, microtiter wells that have been coated with the test organism are exposed to a liquid chemical germicide, and then screened for light production from the surviving bacteria. Exposure to 0.8% phenol causes a reduction in cell numbers of 3 to 10-fold, while exposure to 2% glutaraldehyde shows greater than a 5 log reduction. Since this method does not require outgrowth of the surviving cells, results are obtained in 24 hours, as opposed to 3-4 weeks for the EPA Tuberculocidal Activity Test Method and 2-3 months for the AOAC method 965.12.