Characterization is given of a new parasite, Leishmania equatoriensis sp.n. wich was isolated from the viscera of a sloth (Choloepus hoffmanni) and a squirrel (Sciurus granatensis), captured in humid tropical forest onthe Pacific Coast of Ecuador. Data based on biological and molecular criteria, as well as numerical zymotaxonomical analysis, indicate that this parasite is a new species of the L. brasiliensis complex. L. equatoriensis is cleary distinguishable form all other known species within this complex, using the following molecular criteria: reactivity patterns with specific monoclonal antibodies, isoenzyme electrophoresis, and restriction-endonuclease fragment patterns of kinetoplast DNA (k-DNA).