Healthy Colombian adult volunteers with no history of leishmaniasis were evaluated for evidence of previous subclinical infection with Leishmania based on the Montenegro skin test (MST). Twelve MST-positive subjects were enrolled in an open-label, uncontrolled clinical trial (the "MST-positive trial") and received three injections of the LEISH-F1+MPL-SE vaccine (consisting of 10 microg recombinant Leishmania polyprotein LEISH-F1 antigen [TSA+LmSTI1+LeIF]+25 microg MPL-SE adjuvant). Sixty-eight MST-negative subjects were enrolled in a randomized, double-blind, controlled trial (the "MST-negative trial") and were randomly assigned to receive three injections of either the vaccine (n=34), 10 microg LEISH-F1 protein alone (n=17), or saline placebo (n=17). In both trials, the study injections were given subcutaneously on Days 0, 28, and 56, and subjects were followed for safety and immunological endpoints. The LEISH-F1+MPL-SE vaccine was safe and well tolerated in MST-positive and MST-negative subjects. In both trials, an IFN-gamma response to the LEISH-F1 antigen at Day 84 was observed in more than half of the vaccine recipients. In the MST-negative trial, the IFN-gamma response was significantly more frequent and of greater magnitude in vaccine recipients than in protein-alone or placebo recipients. An IgG antibody response to LEISH-F1 was observed in all vaccine recipients. In both trials, delayed-type hypersensitivity (DTH) to LEISH-F1 was observed in most of the vaccine recipients. In the MST-negative trial, DTH was significantly higher in vaccine than placebo recipients. These clinical trials of the first defined vaccine for leishmaniasis show that the LEISH-F1+MPL-SE vaccine is safe and immunogenic in healthy subjects with and without evidence of previous subclinical infection with Leishmania.