We describe the inhibitory effect of one type of lapachone derived from lapachol, a natural naphtoquinone extracted from the american tree Tabebuia, on the enzyme Na⁺, K⁺ ATPase. This compound shows a diversity of useful biological activities as against several lines of cancer cells and against HIV virus. Drugs inhibiting this ATPase could be important in the treatment of heart failure, if they are less toxic than the commonly used cardiac glycosides. Na⁺, K⁺ ATPase were prepared from guinea pig brain (that contains mainly α₂ and α₃ isoforms) and kidney (containing predominantly α₁) by a modified Jorgensen's method. Freeze-dried preparations had specific activities around 100μmol of product formed/h/mg of protein. Ouabain insensitive activity was almost absent. Enzyme assays (final volume 120μL) were performed in microtiter plates as already described by us (Infection and Immunity, 65:4, 1997). The medium contained a volume of enzyme preparation corresponding to an activity of 0.19-0.21 m mol of product formed/h per assay and concentrations of a -lapachone from 0 to 2,5 mM. The drug was dissolved in DMSO. Controls contained only adequate amounts of DMSO. Results showed that brain isoforms are much more sensitive to the drug than the kidney enzyme. In a concentration of 1,7 mM α-lapachone inhibited 28% the renal enzyme and 58% the brain enzyme. The mechanism of action involved is now being investigated. In conclusion, this drug, that inhibits the enzyme much less dramatically than cardiac glycosides (which are extremely toxic), may prove useful in the future as a therapeutic tool.