Leishmania amazonensis promastigotes are known to express furosemide (Lasix®)-sensitive P-type membrane Na+-ATPase. In the present study, furosemide activity was studied in intracellular amastigotes and infected BALB/c mice to investigate its efficacy in cutaneous leishmaniasis (CL). Intracellular parasites, but not macrophages, were found to be sensitive to killing by furosemide (IC50 = 87 m vs CC50 1000 m, respectively). Although furosemide did not induce nitric oxide production or intracellular pH changes in infected macrophages, it led to a significant reactive oxygen species (ROS) burst. Freshly isolated tissue parasites expressed a high degree of Na+-ATPase activity that decreased with culture, indicative of a higher enzyme expression in amastigotes than in promastigotes. Both intraperitoneal and oral treatment of L. amazonensis-infected mice with furosemide dosages equivalent to that prescribed as a diuretic significantly reduced the parasite's growth compared with the situation in untreated mice. Combination with oral furosemide increased the efficacy and safety of intraperitoneal treatment with sodium stibogluconate (SSG). To summarize, furosemide control of intracellular leishmanial growth by means of parasite Na+-ATPase inhibition, and macrophage ROS activation may help explain its sole and SSG-combined therapeutic effect against murine CL.