Mycobacteria, endogenous respiration, phosphate metabolism, ethambutol, mode of action 1. Under conditions of the endogenous respiration the mode of action of ethambutol on mycobacteria was studied. Oxygen uptake, 32 P-incorporation and changes in the phosphate-content were measured as parameters for the behaviour of the metabolism.2. In order to localize the first step of inhibition the radioactive isotope was applicated in a fixed chronological order and a fractionation in acid-soluble fraction, polyphosphate-, RNA-and DNA-fraction was carried out.3. Proportional to the incubation periode the 32 P was incorporated into the acid-soluble fraction, into the polyphosphate-and into the RNA-fraction in about the same proportions. The content in the DNA-fraction is nearly 200-times lower than in the RNA-fraction.4. The main part of the phosphate is localized in the RNA (~60%). Only in the begin of the incubation periode the part of the acid-soluble fraction is higher than that of the polyphosphate fraction.5. The main part of the phosphate, transferred from the culture medium into the bacteries, was found in the polyphosphate fraction. During the incubation periode the phosphate-content in the RNA-fraction remains nearly unchanged. After decreasing to the begin the same was found for the acid-soluble fraction.6. The specific activity of the polyphosphate fraction rises not continuously, according to the changes of the polyphosphate-content. Depending on the incubation periode the occured changes in the relations of the single fractions are similar in both the control-and the Ethambutol-trial.7. In contrary to the results of other authors these investigations showed the same degree of inhibition of the phosphate metabolism in the acidsoluble fraction, the polyphosphate and the RNAfraction.8. Under conditions of the endogenous respiration ethambutol is inhibiting the phosphorylation of specific compounds of the intermediar-metabolism in the acid-soluble fraction or their further reactions. On the other side the changed incorporation of the 32 P can be explained with a alterated permeability of the cell wall of the bacteria.