A method for the rapid screening of drugs targeting the bioenergetic metabolism of Leishmania spp. was developed. The system is based on the monitoring of changes in the intracellular ATP levels of Leishmania donovani promastigotes that occur in vivo, as assessed by the luminescence produced by parasites transfected with a cytoplasmic form of Phothinus pyralis luciferase and incubated with free-membrane permeable D-luciferin analogue D-luciferin-[1-(4,5-dimethoxy-2-nitrophenyl) ethyl ester]. A significant correlation was obtained between the rapid inhibition of luminescence with parasite proliferation and the dissipation of changes in mitochondrial membrane potential (DeltaPsi(m)) produced by buparvaquone or plumbagin, two leishmanicidal inhibitors of oxidative phosphorylation. To further validate this test, a screen of 14 standard leishmanicidal drugs, using a 50 microM cutoff, was carried out. Despite its semiquantitative properties and restriction to the promastigote stage, this test compares favorably with other bioenergetic parameters with respect to time and cell number requirements for the screening of drugs that affect mitochondrial activity.