Two recombinant barley cystatins, HvCPI5 and HvCPI6, have been tested in vitro against promastigotes and intracellular amastigotes of Leishmania infantum in the J774 monocytic cell line. Toxicity of cystatins for J774 cells was also determined. In addition, a comparison between direct counts of intracellular amastigotes and quantitation of burden by Q-PCR was carried out. Low concentrations (2 microM) from both cystatins were unable to inhibit promastigote replication. HvCPI5 was toxic for mammalian cells; 0.1 microM reduced by more than 50% the cell viability. On the contrary, HvCPI6 did not exhibit any toxicity for J774 cells up to 6 microM and inhibited the intracellular amastigote multiplication. Dose-response analysis showed that 4.8 microM HvCPI6 reduced by >90% the intracellular parasite load and had an approximate IC(50) value of 1.5 microM. Comparable results were obtained by direct counting of intracellular amastigotes and Q-PCR. Results point towards the direct inhibition of amastigote multiplication by HvCPI6 and the interest of this recombinant cystatin in the chemotherapy of leishmaniasis.