The mycobacillin-sensitive Aspergillus niger strain G3Br and resistant mutants of it did not show any differences in their total lipid content, although the amounts of phospholipids and sterols, particularly phosphatidylcholine and cholesterol, were lower in resistant cells. Mycobacillin resistance was accompanied by an increase in the phase-transition temperature of plasma membrane preparations. When exposed to mycobacillin, resistant and sensitive cells did not differ qualitatively with respect to most released materials (lysine, proline, P(μ) Na+, K+, Ca2+); however, the release of ATP was completely inhibited in resistant cells unless they were exposed to concentrations of mycobacillin exceeding their respective MIC value. Resistant cells, under steady-state conditions, displayed greater uptake and release of the same specific materials - except ATP - as sensitive cells did under similar conditions. Thus release and uptake of those materials except ATP are not implicated in the mode of action of mycobacillin. The inhibiting action of mycobacillin (at concentrations higher than the MIC) on sensitive or resistant cells was completely antagonized by ATP (which did not form any complex with mycobacillin) but not by any of the releasable components, either alone or in combination. This observation, coupled with the authors' recent findings on ATP release, indicates that the fungistatic action of mycobacillin is due to excessive ATP release, leading to energy starvation. Interestingly, ATP release during the first 2 h of incubation with mycobacillin was minimal, but increased to over 96% during the next 48 h. Release and uptake of ATP via liposomes, prepared with lipid and protein isolated from membranes of the mycobacillin-sensitive parent and resistant mutants, showed that mycobacillin action could be inhibited either by resistant protein or by resistant lipid. The mycobacillin target appears to be a lipid-protein site on the membrane of sensitive A. niger G3Br.