Although the naphthoquinone, 7-methyljuglone (7-MJ), is active against Mycobacterium tuberculosis (MTB) in vitro, neither the cellular site nor mechanism of anti-mycobacterial action of this agent has been identified. The primary objective of the current study was to investigate the mycobacterial outer membrane as a potential target of 7-MJ by measuring the effects of this agent (0.023 - 1.5 mg/L) on microbial ATP levels and uptake of K+. Methods: Bioluminescence and radiometric (uptake of 86Rb+) procedures were used to assay microbial ATP levels and K+ trans-port respectively. Results: Exposure of MTB (strain H37Rv) to 7-MJ for 60 min resulted in dose-related decreases in both microbial ATP levels and uptake of 86Rb+ which achieved statistical significance (P < 0.05) at concentrations of 0.4 and 0.1 mg/L respectively. Conclusions: These observations are compatible with the mycobacterial membrane as being the putative site of action of 7-MJ, targeting microbial energy metabolism and K+ transport.